Metabolic engineering of arabinose-fermenting yeast cells

ABSTRACT

The invention relates to an eukaryotic cell expressing nucleotide sequences encoding the ara A, ara B and ara D enzymes whereby the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol. Optionally, the eukaryotic cell is also able to convert xylose into ethanol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/442,013, filed Jun. 12, 2009, which is a U.S. national phase of Int'lAppln. No. PCT/NL2007/00246, filed Oct. 1, 2007, which designated theU.S. and claims priority to Appln. No. EP 06121633.9 filed Oct. 2, 2006,and Appln. No. U.S. 60/848,357, filed Oct. 2, 2006; the entire contentsof each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to an eukaryotic cell having the ability to useL-arabinose and/or to convert L-arabinose into L-ribulose, and/orxylulose 5-phosphate and/or into a desired fermentation product and to aprocess for producing a fermentation product wherein this cell is used.

BACKGROUND OF THE INVENTION

Fuel ethanol is acknowledged as a valuable alternative to fossil fuels.Economically viable ethanol production from the hemicellulose fractionof plant biomass requires the simultaneous fermentative conversion ofboth pentoses and hexoses at comparable rates and with high yields.Yeasts, in particular Saccharomyces spp., are the most appropriatecandidates for this process since they can grow and ferment fast onhexoses, both aerobically and anaerobically. Furthermore they are muchmore resistant to the toxic environment of lignocellulose hydrolysatesthan (genetically modified) bacteria.

EP 1 499 708 describes a process for making S. cerevisiae strains ableto produce ethanol from L-arabinose. These strains were modified byintroducing the araA (L-arabinose isomerase) gene from Bacillussubtilis, the araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase)genes from Escherichia coli. Furthermore, these strains were eithercarrying additional mutations in their genome or overexpressing a TAL1(transaldolase) gene. However, these strains have several drawbacks.They ferment arabinose in oxygen limited conditions. In addition, theyhave a low ethanol production rate of 0.05 g.g⁻¹.h⁻¹ (Becker and Boles,2003). Furthermore, these strains are not able to use L-arabinose underanaerobic conditions. Finally, these S. cerevisiae strains have a wildtype background, therefore they can not be used to co-ferment several C5sugars.

WO 03/062430 and WO 06/009434 disclose yeast strains able to convertxylose into ethanol. These yeast strains are able to directly isomerisexylose into xylulose.

Still, there is a need for alternative strains for producing ethanol,which perform better and are more robust and resistant to relativelyharsh production conditions.

DESCRIPTION OF THE FIGURES

FIG. 1. Plasmid maps of pRW231 and pRW243.

FIG. 2. Growth pattern of shake flask cultivations of strain RWB219 (◯)and IMS0001 () in synthetic medium containing 0.5% galactose (A) and0.1% galactose+2% L-arabinose (B). Cultures were grown for 72 hours insynthetic medium with galactose (A) and then transferred to syntheticmedium with galactose and arabinose (B). Growth was determined bymeasuring the OD₆₆₀.

FIG. 3. Growth rate during serial transfers of S. cerevisiae IMS0001 inshake flask cultures containing synthetic medium with 2% (w/v)L-arabinose. Each datapoint represents the growth rate estimated fromthe OD₆₆₀ measured during (exponential) growth. The closed and opencircles represent duplicate serial transfer experiments.

FIG. 4. Growth rate during an anaerobic SBR fermentation of S.cerevisiae IMS0001 in synthetic medium with 2% (w/v) L-arabinose. Eachdatapoint represents the growth rate estimated from the CO₂ profile(solid line) during exponential growth.

FIG. 5. Sugar consumption and product formation during anaerobic batchfermentations of strain IMS0002. The fermentations were performed in 1synthetic medium supplemented with: 20 gl⁻¹ arabinose (A); 20 gl⁻¹glucose and 20 gl⁻¹ arabinose (B); 30 gl⁻¹ glucose, 15 gl⁻¹ xylose, and15 gl⁻¹ arabinose (C); Sugar consumption and product formation duringanaerobic batch fermentations with a mixture of strains IMS0002 andRWB218. The fermentations were performed in 1 liter of synthetic mediumsupplemented with 30 gl⁻¹ glucose, 15 gl⁻¹ xylose, and 15 gl⁻¹ arabinose(D). Symbols: glucose (); xylose (◯); arabinose (▪); ethanol calculatedfrom cumulative CO₂ production (□); ethanol measured by HPLC (▴);cumulative CO₂ production (Δ); xylitol (▾)

FIG. 6. Sugar consumption and product formation during an anaerobicbatch fermentation of strain IMS0002 cells selected for anaerobic growthon xylose. The fermentation was performed in 1 liter of synthetic mediumsupplemented with 20 gl⁻¹ xylose and 20 gl⁻¹ arabinose. Symbols: xylose(◯); arabinose (▪); ethanol measured by HPLC (▴); cumulative CO₂production (Δ); xylitol (▾).

FIG. 7. Sugar consumption and product formation during an anaerobicbatch fermentation of strain IMS0003. The fermentation was performed in1 liter of synthetic medium supplemented with: 30 gl⁻¹ glucose, 15 gl⁻¹xylose, and 15 gl⁻¹ arabinose. Symbols: glucose (); xylose (◯);arabinose (▪); ethanol calculated from cumulative CO₂ production (□);ethanol measured by HPLC (▴); cumulative CO₂ production (Δ);

DESCRIPTION OF THE INVENTION Eukaryotic Cell

In a first aspect, the invention relates to a eukaryotic cell capable ofexpressing the following nucleotide sequences, whereby the expression ofthese nucleotide sequences confers on the cell the ability to useL-arabinose and/or to convert L-arabinose into L-ribulose, and/orxylulose 5-phosphate and/or into a desired fermentation product such asethanol:

-   -   (a) a nucleotide sequence encoding an arabinose isomerase        (araA), wherein said nucleotide sequence is selected from the        group consisting of:        -   (i) nucleotide sequences encoding an araA, said araA            comprising an amino acid sequence that has at least 55%            sequence identity with the amino acid sequence of SEQ ID            NO:1.        -   (ii) nucleotide sequences comprising a nucleotide sequence            that has at least 60% sequence identity with the nucleotide            sequence of SEQ ID NO:2.        -   (iii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii);        -   (iv) nucleotide sequences the sequences of which differ from            the sequence of a nucleic acid molecule of (iii) due to the            degeneracy of the genetic code,    -   (b) a nucleotide sequence encoding a L-ribulokinase (araB),        wherein said nucleotide sequence is selected from the group        consisting of:        -   (i) nucleotide sequences encoding an araB, said araB            comprising an amino acid sequence that has at least 20%            sequence identity with the amino acid sequence of SEQ ID            NO:3.        -   (ii) nucleotide sequences comprising a nucleotide sequence            that has at least 50% sequence identity with the nucleotide            sequence of SEQ ID NO:4.        -   (iii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii);        -   (iv) nucleotide sequences the sequences of which differ from            the sequence of a nucleic acid molecule of (iii) due to the            degeneracy of the genetic code,    -   (c) a nucleotide sequence encoding an L-ribulose-5-P-4-epimerase        (araD), wherein said nucleotide sequence is selected from the        group consisting of:        -   (i) nucleotide sequences encoding an araD, said araD            comprising an amino acid sequence that has at least 60%            sequence identity with the amino acid sequence of SEQ ID            NO:5.        -   (ii) nucleotide sequences comprising a nucleotide sequence            that has at least 60% sequence identity with the nucleotide            sequence of SEQ ID NO:6.        -   (iii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii);        -   (iv) nucleotide sequences the sequences of which differ from            the sequence of a nucleic acid molecule of (iii) due to the            degeneracy of the genetic code.            A preferred embodiment relates to an eukaryotic cell capable            of expressing the following nucleotide sequences, whereby            the expression of these nucleotide sequences confers on the            cell the ability to use L-arabinose and/or to convert            L-arabinose into L-ribulose, and/or xylulose 5-phosphate            and/or into a desired fermentation product such as ethanol:    -   (a) a nucleotide sequence encoding an arabinose isomerase        (araA), wherein said nucleotide sequence is selected from the        group consisting of:        -   (i) nucleotide sequences comprising a nucleotide sequence            that has at least 60% sequence identity with the nucleotide            sequence of SEQ ID NO:2,        -   (ii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i);        -   (iii) nucleotide sequences the sequences of which differ            from the sequence of a nucleic acid molecule of (ii) due to            the degeneracy of the genetic code,    -   (b) a nucleotide sequence encoding a L-ribulokinase (araB),        wherein said nucleotide sequence is selected from the group        consisting of:        -   (i) nucleotide sequences encoding an araB, said araB            comprising an amino acid sequence that has at least 20%            sequence identity with the amino acid sequence of SEQ ID            NO:3.        -   (ii) nucleotide sequences comprising a nucleotide sequence            that has at least 50% sequence identity with the nucleotide            sequence of SEQ ID NO:4.        -   (iii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii);        -   (iv) nucleotide sequences the sequences of which differ from            the sequence of a nucleic acid molecule of (iii) due to the            degeneracy of the genetic code,    -   (c) a nucleotide sequence encoding an L-ribulose-5-P-4-epimerase        (araD), wherein said nucleotide sequence is selected from the        group consisting of:        -   (i) nucleotide sequences encoding an araD, said araD            comprising an amino acid sequence that has at least 60%            sequence identity with the amino acid sequence of SEQ ID            NO:5.        -   (ii) nucleotide sequences comprising a nucleotide sequence            that has at least 60% sequence identity with the nucleotide            sequence of SEQ ID NO:6.        -   (iii) nucleotide sequences the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii);        -   (iv) nucleotide sequences the sequences of which differ from            the sequence of a nucleic acid molecule of (iii) due to the            degeneracy of the genetic code.

Sequence Identity and Similarity

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences. “Similarity” between two amino acidsequences is determined by comparing the amino acid sequence and itsconserved amino acid substitutes of one polypeptide to the sequence of asecond polypeptide. “Identity” and “similarity” can be readilycalculated by various methods, known to those skilled in the art.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the BestFit, BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990),publicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894). A most preferredalgorithm used is EMBOSS (http://www.ebi.ac.uk/emboss/align). Preferredparameters for amino acid sequences comparison using EMBOSS are gap open10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleicacid sequences comparison using EMBOSS are gap open 10.0, gap extend0.5, DNA full matrix (DNA identity matrix).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

Hybridising Nucleic Acid Sequences

Nucleotide sequences encoding the enzymes expressed in the cell of theinvention may also be defined by their capability to hybridise with thenucleotide sequences of SEQ ID NO.'s 2, 4, 6, 8, 16, 18, 20, 22, 24, 26,28, 30 respectively, under moderate, or preferably under stringenthybridisation conditions. Stringent hybridisation conditions are hereindefined as conditions that allow a nucleic acid sequence of at leastabout 25, preferably about 50 nucleotides, 75 or 100 and most preferablyof about 200 or more nucleotides, to hybridise at a temperature of about65° C. in a solution comprising about 1 M salt, preferably 6×SSC or anyother solution having a comparable ionic strength, and washing at 65° C.in a solution comprising about 0.1 M salt, or less, preferably 0.2×SSCor any other solution having a comparable ionic strength. Preferably,the hybridisation is performed overnight, i.e. at least for 10 hours andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having about 90% or more sequenceidentity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

AraA

A preferred nucleotide sequence encoding a arabinose isomerase (araA)expressed in the cell of the invention is selected from the groupconsisting of:

-   (a) nucleotide sequences encoding an araA polypeptide said araA    comprising an amino acid sequence that has at least 55, 60, 65, 70,    75, 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino    acid sequence of SEQ ID NO. 1;-   (b) nucleotide sequences comprising a nucleotide sequence that has    at least 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity with    the nucleotide sequence of SEQ ID NO. 2;-   (c) nucleotide sequences the complementary strand of which    hybridises to a nucleic acid molecule sequence of (a) or (b);-   (d) nucleotide sequences the sequence of which differ from the    sequence of a nucleic acid molecule of (c) due to the degeneracy of    the genetic code.    The nucleotide sequence encoding an araA may encode either a    prokaryotic or an eukaryotic araA, i.e. an araA with an amino acid    sequence that is identical to that of an araA that naturally occurs    in the prokaryotic or eukaryotic organism. The present inventors    have found that the ability of a particular araA to confer to a    eukaryotic host cell the ability to use arabinose and/or to convert    arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a    desired fermentation product such as ethanol when co-expressed with    araB and araD does not depend so much on whether the araA is of    prokaryotic or eukaryotic origin. Rather this depends on the    relatedness of the araA's amino acid sequence to that of the    sequence SEQ ID NO. 1.

AraB

A preferred nucleotide sequence encoding a L-ribulokinase (AraB)expressed in the cell of the invention is selected from the groupconsisting of:

-   -   (a) nucleotide sequences encoding a polypeptide comprising an        amino acid sequence that has at least 20, 25, 30, 35, 40, 45,        50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence        identity with the amino acid sequence of SEQ ID NO. 3;    -   (b) nucleotide sequences comprising a nucleotide sequence that        has at least 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence        identity with the nucleotide sequence of SEQ ID NO.4;    -   (c) nucleotide sequences the complementary strand of which        hybridises to a nucleic acid molecule sequence of (a) or (b);    -   (d) nucleotide sequences the sequence of which differ from the        sequence of a nucleic acid molecule of (c) due to the degeneracy        of the genetic code.        The nucleotide sequence encoding an araB may encode either a        prokaryotic or an eukaryotic araB, i.e. an araB with an amino        acid sequence that is identical to that of a araB that naturally        occurs in the prokaryotic or eukaryotic organism. The present        inventors have found that the ability of a particular araB to        confer to a eukaryotic host cell the ability to use arabinose        and/or to convert arabinose into L-ribulose, and/or xylulose        5-phosphate and/or into a desired fermentation product when        co-expressed with araA and araD does not depend so much on        whether the araB is of prokaryotic or eukaryotic origin. Rather        this depends on the relatedness of the araB's amino acid        sequence to that of the sequence SEQ ID NO. 3.

AraD

A preferred nucleotide sequence encoding a L-ribulose-5-P-4-epimerase(araD) expressed in the cell of the invention is selected from the groupconsisting of:

-   -   (e) nucleotide sequences encoding a polypeptide comprising an        amino acid sequence that has at least 60, 65, 70, 75, 80, 85,        90, 95, 97, 98, or 99% sequence identity with the amino acid        sequence of SEQ ID NO. 5;    -   (f) nucleotide sequences comprising a nucleotide sequence that        has at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99%        sequence identity with the nucleotide sequence of SEQ ID NO.6;    -   (g) nucleotide sequences the complementary strand of which        hybridises to a nucleic acid molecule sequence of (a) or (b);    -   (h) nucleotide sequences the sequence of which differs from the        sequence of a nucleic acid molecule of (c) due to the degeneracy        of the genetic code.        The nucleotide sequence encoding an araD may encode either a        prokaryotic or an eukaryotic araD, i.e. an araD with an amino        acid sequence that is identical to that of a araD that naturally        occurs in the prokaryotic or eukaryotic organism. The present        inventors have found that the ability of a particular araD to        confer to a eukaryotic host cell the ability to use arabinose        and/or to convert arabinose into L-ribulose, and/or xylulose        5-phosphate and/or into a desired fermentation product when        co-expressed with araA and araB does not depend so much on        whether the araD is of prokaryotic or eukaryotic origin. Rather        this depends on the relatedness of the araD's amino acid        sequence to that of the sequence SEQ ID NO. 5.

Surprisingly, the codon bias index indicated that expression of theLactobacillus plantarum araA, araB and araD genes were more favorablefor expression in yeast than the prokaryolic araA, araB and araD genesdescribed in EP 1 499 708.

It is to be noted that L. plantarum is a Generally Regarded As Safe(GRAS) organism, which is recognized as safe by food registrationauthorities. Therefore, a preferred nucleotide sequence encodes an araA,araB or araD respectively having an amino acid sequence that is relatedto the sequences SEQ ID NO: 1, 3, or 5 respectively as defined above. Apreferred nucleotide sequence encodes a fungal araA, araB or araDrespectively (e.g. from a Basidiomycete), more preferably an araA, araBor araD respectively from an anaerobic fungus, e.g. an anaerobic fungusthat belongs to the families Neocallimastix, Caecomyces, Piromyces,Orpinomyces, or Ruminomyces. Alternatively, a preferred nucleotidesequence encodes a bacterial araA, araB or araD respectively, preferablyfrom a Gram-positive bacterium, more preferably from the genusLactobacillus, most preferably from Lactobacillus plantarum species.Preferably, one, two or three or the araA, araB and araD nucleotidesequences originate from a Lactobacillus genus, more preferably aLactobacillus plantarum species. The bacterial araA expressed in thecell of the invention is not the Bacillus subtilis araA disclosed in EP1 499 708 and given as SEQ ID NO:9. SEQ ID NO:10 represents thenucleotide acid sequence coding for SEQ ID NO:9. The bacterial araB andaraD expressed in the cell of the invention are not the ones ofEscherichia coli (E. coli) as disclosed in EP 1 499 708 and given as SEQID NO: 11 and SEQ ID NO:13. SEQ ID NO: 12 represents the nucleotide acidsequence coding for SEQ ID NO:11. SEQ ID NO:14 represents the nucleotideacid sequence coding for SEQ ID NO:13.

To increase the likelihood that the (bacterial) araA, araB and araDenzymes respectively are expressed in active form in a eukaryotic hostcell of the invention such as yeast, the corresponding encodingnucleotide sequence may be adapted to optimise its codon usage to thatof the chosen eukaryotic host cell. The adaptiveness of a nucleotidesequence encoding the araA, araB, and araD enzymes (or other enzymes ofthe invention, see below) to the codon usage of the chosen host cell maybe expressed as codon adaptation index (CAI). The codon adaptation indexis herein defined as a measurement of the relative adaptiveness of thecodon usage of a gene towards the codon usage of highly expressed genes.The relative adaptiveness (w) of each codon is the ratio of the usage ofeach codon, to that of the most abundant codon for the same amino acid.The CAI index is defined as the geometric mean of these relativeadaptiveness values. Non-synonymous codons and termination codons(dependent on genetic code) are excluded. CAI values range from 0 to 1,with higher values indicating a higher proportion of the most abundantcodons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295;also see: Jansen et al., 2003, Nucleic Acids Res. 31(8):2242-51). Anadapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3,0.4, 0.5, 0.6 or 0.7.

In a preferred embodiment, expression of the nucleotide sequencesencoding an ara A, an ara B and an ara D as defined earlier hereinconfers to the cell the ability to use L-arabinose and/or to convert itinto L-ribulose, and/or xylulose 5-phosphate. Without wishing to bebound by any theory, L-arabinose is expected to be first converted intoL-ribulose, which is subsequently converted into xylulose 5-phosphatewhich is the main molecule entering the pentose phosphate pathway. Inthe context of the invention, “using L-arabinose” preferably means thatthe optical density measured at 660 nm (OD₆₆₀) of transformed cellscultured under aerobic or anaerobic conditions in the presence of atleast 0.5% L-arabinose during at least 20 days is increased fromapproximately 0.5 till 1.0 or more. More preferably, the OD₆₆₀ isincreased from 0.5 till 1.5 or more. More preferably, the cells arecultured in the presence of at least 1%, at least 1.5%, at least 2%L-arabinose. Most preferably, the cells are cultured in the presence ofapproximately 2% L-arabinose.In the context of the invention, a cell is able “to convert L-arabinoseinto L-ribulose” when detectable amounts of L-ribulose are detected incells cultured under aerobic or anaerobic conditions in the presence ofL-arabinose (same preferred concentrations as in previous paragraph)during at least 20 days using a suitable assay. Preferably the assay isHPLC for L-ribulose.In the context of the invention, a cell is able “to convert L-arabinoseinto xylulose 5-phosphate” when an increase of at least 2% of xylulose5-phosphate is detected in cells cultured under aerobic or anaerobicconditions in the presence of L-arabinose (same preferred concentrationsas in previous paragraph) during at least 20 days using a suitableassay. Preferably, an HPCL-based assay for xylulose 5-phosphate has beendescribed in Zaldivar J., et al ((2002), Appl. Microbiol. Biotechnol.,59:436-442). This assay is briefly described in the experimental part.More preferably, the increase is of at least 5%, 10%, 15%, 20%, 25% ormore.In another preferred embodiment, expression of the nucleotide sequencesencoding an ara A, ara B and ara D as defined earlier herein confers tothe cell the ability to convert L-arabinose into a desired fermentationproduct when cultured under aerobic or anaerobic conditions in thepresence of L-arabinose (same preferred concentrations as in previousparagraph) during at least one month till one year. More preferably, acell is able to convert L-arabinose into a desired fermentation productwhen detectable amounts of a desired fermentation product are detectedusing a suitable assay and when the cells are cultured under theconditions given in previous sentence. Even more preferably, the assayis HPLC. Even more preferably, the fermentation product is ethanol.

A cell for transformation with the nucleotide sequences encoding thearaA, araB, and araD enzymes respectively as described above, preferablyis a host cell capable of active or passive xylose transport into andxylose isomerisation within the cell. The cell preferably is capable ofactive glycolysis. The cell may further contain an endogenous pentosephosphate pathway and may contain endogenous xylulose kinase activity sothat xylulose isomerised from xylose may be metabolised to pyruvate. Thecell further preferably contains enzymes for conversion of pyruvate to adesired fermentation product such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotic or a cephalosporin.The cell may be made capable of producing butanol by introduction of oneor more genes of the butanol pathway as disclosed in WO2007/041269.

A preferred cell is naturally capable of alcoholic fermentation,preferably, anaerobic alcoholic fermentation. The host cell furtherpreferably has a high tolerance to ethanol, a high tolerance to low pH(i.e. capable of growth at a pH lower than 5, 4, 3, or 2,5) and towardsorganic acids like lactic acid, acetic acid or formic acid and sugardegradation products such as furfural and hydroxy-methylfurfural, and ahigh tolerance to elevated temperatures. Any of these characteristics oractivities of the host cell may be naturally present in the host cell ormay be introduced or modified through genetic selection or by geneticmodification. A suitable host cell is a eukaryotic microorganism likee.g. a fungus, however, most suitable as host cell are yeasts orfilamentous fungi.

Yeasts are herein defined as eukaryotic microorganisms and include allspecies of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In:Introductory Mycology, John Wiley & Sons, Inc., New York) thatpredominantly grow in unicellular form. Yeasts may either grow bybudding of a unicellular thallus or may grow by fission of the organism.Preferred yeasts as host cells belong to one of the generaSaccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces,Hansenula, Kloeckera, Schwanniomyces, or Yarrowia. Preferably the yeastis capable of anaerobic fermentation, more preferably anaerobicalcoholic fermentation.

Filamentous fungi are herein defined as eukaryotic microorganisms thatinclude all filamentous forms of the subdivision Eumycotina. These fungiare characterized by a vegetative mycelium composed of chitin,cellulose, and other complex polysaccharides. The filamentous fungi ofthe present invention are morphologically, physiologically, andgenetically distinct from yeasts. Vegetative growth by filamentous fungiis by hyphal elongation and carbon catabolism of most filamentous fungiis obligately aerobic. Preferred filamentous fungi as host cells belongto one of the genera Aspergillus, Trichoderma, Humicola, Acremonium,Fusarium, or Penicillium.

Over the years suggestions have been made for the introduction ofvarious organisms for the production of bio-ethanol from crop sugars. Inpractice, however, all major bio-ethanol production processes havecontinued to use the yeasts of the genus Saccharomyces as ethanolproducer. This is due to the many attractive features of Saccharomycesspecies for industrial processes, i.e., a high acid-, ethanol- andosmo-tolerance, capability of anaerobic growth, and of course its highalcoholic fermentative capacity. Preferred yeast species as host cellsinclude S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum,S. diastaticus, K. lactis, K. marxianus, K. fragilis.

In a preferred embodiment, the host cell of the invention is a host cellthat has been transformed with a nucleic acid construct comprising thenucleotide sequence encoding the araA, araB, and araD enzymes as definedabove. In one more preferred embodiment, the host cell is co-transformedwith three nucleic acid constructs, each nucleic acid constructcomprising the nucleotide sequence encoding araA, araB or araD. Thenucleic acid construct comprising the araA, araB, and/or araD codingsequence is capable of expression of the araA, araB, and/or araD enzymesin the host cell. To this end the nucleic acid construct may beconstructed as described in e.g. WO 03/0624430. The host cell maycomprise a single but preferably comprises multiple copies of eachnucleic acid construct. The nucleic acid construct may be maintainedepisomally and thus comprise a sequence for autonomous replication, suchas an ARS sequence. Suitable episomal nucleic acid constructs may e.g.be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology9:968-975) plasmids. Preferably, however, each nucleic acid construct isintegrated in one or more copies into the genome of the host cell.Integration into the host cell's genome may occur at random byillegitimate recombination but preferably nucleic acid construct isintegrated into the host cell's genome by homologous recombination as iswell known in the art of fungal molecular genetics (see e.g. WO90/14423, EP-A-0 481 008, EP-A-0 635 574 and U.S. Pat. No. 6,265,186).Accordingly, in a more preferred embodiment, the cell of the inventioncomprises a nucleic acid construct comprising the araA, araB, and/oraraD coding sequence and is capable of expression of the araA, araB,and/or araD enzymes. In an even more preferred embodiment, the araA,araB, and/or araD coding sequences are each operably linked to apromoter that causes sufficient expression of the correspondingnucleotide sequences in a cell to confer to the cell the ability to useL-arabinose, and/or to convert L-arabinose into L-ribulose, and/orxylulose 5-phosphate. Preferably the cell is a yeast cell. Accordingly,in a further aspect, the invention also encompasses a nucleic acidconstruct as earlier outlined herein. Preferably, a nucleic acidconstruct comprises a nucleic acid sequence encoding an araA, araBand/or araD. Nucleic acid sequences encoding an araA, araB, or araD havebeen all earlier defined herein. Even more preferably, the expression ofthe corresponding nucleotide sequences in a cell confer to the cell theability to convert L-arabinose into a desired fermentation product asdefined later herein. In an even more preferred embodiment, thefermentation product is ethanol. Even more preferably, the cell is ayeast cell.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence)in a functional relationship. A nucleic acid sequence is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thecoding sequence. Operably linked means that the nucleic acid sequencesbeing linked are typically contiguous and, where necessary to join twoprotein coding regions, contiguous and in reading frame.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

The promoter that could be used to achieve the expression of thenucleotide sequences coding for araA, araB and/or araD may be not nativeto the nucleotide sequence coding for the enzyme to be expressed, i.e. apromoter that is heterologous to the nucleotide sequence (codingsequence) to which it is operably linked. Although the promoterpreferably is heterologous to the coding sequence to which it isoperably linked, it is also preferred that the promoter is homologous,i.e. endogenous to the host cell. Preferably the heterologous promoter(to the nucleotide sequence) is capable of producing a higher steadystate level of the transcript comprising the coding sequence (or iscapable of producing more transcript molecules, i.e. mRNA molecules, perunit of time) than is the promoter that is native to the codingsequence, preferably under conditions where arabinose, or arabinose andglucose, or xylose and arabinose or xylose and arabinose and glucose areavailable as carbon sources, more preferably as major carbon sources(i.e. more than 50% of the available carbon source consists ofarabinose, or arabinose and glucose, or xylose and arabinose or xyloseand arabinose and glucose), most preferably as sole carbon sources.Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters. A preferredpromoter for use in the present invention will in addition beinsensitive to catabolite (glucose) repression and/or will preferablynot require arabinose and/or xylose for induction.

Promotors having these characteristics are widely available and known tothe skilled person. Suitable examples of such promoters include e.g.promoters from glycolytic genes, such as the phosphofructokinase (PPK),triose phosphate isomerase (TPI), glyceraldehyde-3-phosphatedehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK),phosphoglycerate kinase (PGK) promoters from yeasts or filamentousfungi; more details about such promoters from yeast may be found in (WO93/03159). Other useful promoters are ribosomal protein encoding genepromoters, the lactase gene promoter (LAC4), alcohol dehydrogenasepromoters (ADH1, ADH4, and the like), the enolase promoter (ENO), theglucose-6-phosphate isomerase promoter (PGI1, Hauf et al, 2000) or thehexose(glucose) transporter promoter (HXT7) or theglyceraldehyde-3-phosphate dehydrogenase (TDH3). The sequence of thePGI1 promoter is given in SEQ ID NO:51. The sequence of the HXT7promoter is given in SEQ ID NO:52. The sequence of the TDH3 promoter isgiven in SEQ ID NO:49. Other promoters, both constitutive and inducible,and enhancers or upstream activating sequences will be known to those ofskill in the art. The promoters used in the host cells of the inventionmay be modified, if desired, to affect their control characteristics. Apreferred cell of the invention is a eukaryotic cell transformed withthe araA, araB and araD genes of L. plantarum. More preferably, theeukaryotic cell is a yeast cell, even more preferably a S. cerevisiaestrain transformed with the araA, araB and araD genes of L. plantarum.Most preferably, the cell is either CBS120327 or CBS120328 bothdeposited at the CBS Institute (The Netherlands) on Sep. 27, 2006.

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain. Ifhomologous to a host cell, a nucleic acid sequence encoding apolypeptide will typically be operably linked to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. When used toindicate the relatedness of two nucleic acid sequences the term“homologous” means that one single-stranded nucleic acid sequence mayhybridize to a complementary single-stranded nucleic acid sequence. Thedegree of hybridization may depend on a number of factors including theamount of identity between the sequences and the hybridizationconditions such as temperature and salt concentration as earlierpresented. Preferably the region of identity is greater than about 5 bp,more preferably the region of identity is greater than 10 bp.

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein that does not occurnaturally as part of the organism, cell, genome or DNA or RNA sequencein which it is present, or that is found in a cell or location orlocations in the genome or DNA or RNA sequence that differ from that inwhich it is found in nature. Heterologous nucleic acids or proteins arenot endogenous to the cell into which it is introduced, but has beenobtained from another cell or synthetically or recombinantly produced.Generally, though not necessarily, such nucleic acids encode proteinsthat are not normally produced by the cell in which the DNA istranscribed or expressed. Similarly exogenous RNA encodes for proteinsnot normally expressed in the cell in which the exogenous RNA ispresent. Heterologous nucleic acids and proteins may also be referred toas foreign nucleic acids or proteins. Any nucleic acid or protein thatone of skill in the art would recognize as heterologous or foreign tothe cell in which it is expressed is herein encompassed by the termheterologous nucleic acid or protein. The term heterologous also appliesto non-natural combinations of nucleic acid or amino acid sequences,i.e. combinations where at least two of the combined sequences areforeign with respect to each other.

Preferred Eukaryotic Cell Able to Use and/or Convert L-Arabinose andXyloseIn a more preferred embodiment, the cell of the invention that expressesaraA, araB and araD is able to use L-arabinose and/or to convert it intoL-ribulose, and/or xylulose 5-phosphate and/or a desired fermentationproduct as earlier defined herein and additionally exhibits the abilityto use xylose and/or convert xylose into xylulose. The conversion ofxylose into xylulose is preferably a one step isomerisation step (directisomerisation of xylose into xylulose). This type of cell is thereforeable to use both L-arabinose and xylose. “Using” xylose has preferablythe same meaning as “using” L-arabinose as earlier defined herein.Enzyme definitions are as used in WO 06/009434, for xylose isomerase (EC5.3.1.5), xylulose kinase (EC 2.7.1.17), ribulose 5-phosphate epimerase(5.1.3.1), ribulose 5-phosphate isomerase (EC 5.3.1.6), transketolase(EC 2.2.1.1), transaldolase (EC 2.2.1.2), and aldose reductase” (EC1.1.1.21).In a preferred embodiment, the eukaryotic cell of the inventionexpressing araA, araB and araD as earlier defined herein has the abilityof isomerising xylose to xylulose as e.g. described in WO 03/0624430 orin WO 06/009434. The ability of isomerising xylose to xylulose isconferred to the host cell by transformation of the host cell with anucleic acid construct comprising a nucleotide sequence encoding axylose isomerase. The transformed host cell's ability to isomerisexylose into xylulose is the direct isomerisation of xylose to xylulose.This is understood to mean that xylose isomerised into xylulose in asingle reaction catalysed by a xylose isomerase, as opposed to the twostep conversion of xylose into xylulose via a xylitol intermediate ascatalysed by xylose reductase and xylitol dehydrogenase, respectively.

The nucleotide sequence encodes a xylose isomerase that is preferablyexpressed in active form in the transformed host cell of the invention.Thus, expression of the nucleotide sequence in the host cell produces axylose isomerase with a specific activity of at least 10 U xyloseisomerase activity per mg protein at 30° C., preferably at least 20, 25,30, 50, 100, 200, 300 or 500 Upper mg at 30° C. The specific activity ofthe xylose isomerase expressed in the transformed host cell is hereindefined as the amount of xylose isomerase activity units per mg proteinof cell free lysate of the host cell, e.g. a yeast cell free lysate.Determination of the xylose isomerase activity has already beendescribed earlier herein.

Preferably, expression of the nucleotide sequence encoding the xyloseisomerase in the host cell produces a xylose isomerase with a K_(m) forxylose that is less than 50, 40, 30 or 25 mM, more preferably, the K_(m)for xylose is about 20 mM or less.

A preferred nucleotide sequence encoding the xylose isomerase may beselected from the group consisting of:

-   (e) nucleotide sequences encoding a polypeptide comprising an amino    acid sequence that has at least 60, 65, 70, 75, 80, 85, 90, 95, 97,    98, or 99% sequence identity with the amino acid sequence of SEQ ID    NO. 7 or SEQ ID NO:15;-   (f) nucleotide sequences comprising a nucleotide sequence that has    at least 40, 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence    identity with the nucleotide sequence of SEQ ID NO. 8 or SEQ ID    NO:16;-   (g) nucleotide sequences the complementary strand of which    hybridises to a nucleic acid molecule sequence of (a) or (b);-   (h) nucleotide sequences the sequence of which differs from the    sequence of a nucleic acid molecule of (c) due to the degeneracy of    the genetic code.

The nucleotide sequence encoding the xylose isomerase may encode eithera prokaryotic or an eukaryotic xylose isomerase, i.e. a xylose isomerasewith an amino acid sequence that is identical to that of a xyloseisomerase that naturally occurs in the prokaryotic or eukaryoticorganism. The present inventors have found that the ability of aparticular xylose isomerase to confer to a eukaryotic host cell theability to isomerise xylose into xylulose does not depend so much onwhether the isomerase is of prokaryotic or eukaryotic origin. Ratherthis depends on the relatedness of the isomerase's amino acid sequenceto that of the Piromyces sequence (SEQ ID NO. 7). Surprisingly, theeukaryotic Piromyces isomerase is more related to prokaryotic isomerasesthan to other known eukaryotic isomerases. Therefore, a preferrednucleotide sequence encodes a xylose isomerase having an amino acidsequence that is related to the Piromyces sequence as defined above. Apreferred nucleotide sequence encodes a fungal xylose isomerase (e.g.from a Basidiomycete), more preferably a xylose isomerase from ananaerobic fungus, e.g. a xylose isomerase from an anaerobic fungus thatbelongs to the families Neocallimastix, Caecomyces, Piromyces,Orpinomyces, or Ruminomyces. Alternatively, a preferred nucleotidesequence encodes a bacterial xylose isomerase, preferably aGram-negative bacterium, more preferably an isomerase from the classBacteroides, or from the genus Bacteroides, most preferably from B.thetaiotaomicron (SEQ ID NO. 15).

To increase the likelihood that the xylose isomerase is expressed inactive form in a eukaryotic host cell such as yeast, the nucleotidesequence encoding the xylose isomerase may be adapted to optimise itscodon usage to that of the eukaryotic host cell as earlier definedherein.

A host cell for transformation with the nucleotide sequence encoding thexylose isomerase as described above, preferably is a host capable ofactive or passive xylose transport into the cell. The host cellpreferably contains active glycolysis. The host cell may further containan endogenous pentose phosphate pathway and may contain endogenousxylulose kinase activity so that xylulose isomerised from xylose may bemetabolised to pyruvate. The host further preferably contains enzymesfor conversion of pyruvate to a desired fermentation product such asethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, aceticacid, succinic acid, citric acid, malic acid, fumaric acid, an aminoacid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactamantibiotic or a cephalosporin. A preferred host cell is a host cell thatis naturally capable of alcoholic fermentation, preferably, anaerobicalcoholic fermentation. The host cell further preferably has a hightolerance to ethanol, a high tolerance to low pH (i.e. capable of growthat a pH lower than 5, 4, 3, or 2,5) and towards organic acids likelactic acid, acetic acid or formic acid and sugar degradation productssuch as furfural and hydroxy-methylfurfural, and a high tolerance toelevated temperatures. Any of these characteristics or activities of thehost cell may be naturally present in the host cell or may be introducedor modified by genetic modification. A suitable cell is a eukaryoticmicroorganism like e.g. a fungus, however, most suitable as host cellare yeasts or filamentous fungi. Preferred yeasts and filamentous fungihave already been defined herein.

As used herein the wording host cell has the same meaning as cell.

The cell of the invention is preferably transformed with a nucleic acidconstruct comprising the nucleotide sequence encoding the xyloseisomerase. The nucleic acid construct that is preferably used is thesame as the one used comprising the nucleotide sequence encoding araA,araB or araD.

In another preferred embodiment of the invention, the cell of theinvention:

-   -   expressing araA, araB and araD, and exhibiting the ability to        directly isomerise xylose into xylulose, as earlier defined        herein        further comprises a genetic modification that increases the flux        of the pentose phosphate pathway, as described in WO 06/009434.        In particular, the genetic modification causes an increased flux        of the non-oxidative part pentose phosphate pathway. A genetic        modification that causes an increased flux of the non-oxidative        part of the pentose phosphate pathway is herein understood to        mean a modification that increases the flux by at least a factor        1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to the flux in a        strain which is genetically identical except for the genetic        modification causing the increased flux. The flux of the        non-oxidative part of the pentose phosphate pathway may be        measured by growing the modified host on xylose as sole carbon        source, determining the specific xylose consumption rate and        substracting the specific xylitol production rate from the        specific xylose consumption rate, if any xylitol is produced.        However, the flux of the non-oxidative part of the pentose        phosphate pathway is proportional with the growth rate on xylose        as sole carbon source, preferably with the anaerobic growth rate        on xylose as sole carbon source. There is a linear relation        between the growth rate on xylose as sole carbon source        (μ_(max)) and the flux of the non-oxidative part of the pentose        phosphate pathway. The specific xylose consumption rate (Q_(s))        is equal to the growth rate (μ) divided by the yield of biomass        on sugar (Y_(xs)) because the yield of biomass on sugar is        constant (under a given set of conditions: anaerobic, growth        medium, pH, genetic background of the strain, etc.; i.e.        Q_(s)=μ/Y_(xs)). Therefore the increased flux of the        non-oxidative part of the pentose phosphate pathway may be        deduced from the increase in maximum growth rate under these        conditions. In a preferred embodiment, the cell comprises a        genetic modification that increases the flux of the pentose        phosphate pathway and has a specific xylose consumption rate of        at least 346 mg xylose/g biomass/h.

Genetic modifications that increase the flux of the pentose phosphatepathway may be introduced in the host cell in various ways. Theseincluding e.g. achieving higher steady state activity levels of xylulosekinase and/or one or more of the enzymes of the non-oxidative partpentose phosphate pathway and/or a reduced steady state level ofunspecific aldose reductase activity. These changes in steady stateactivity levels may be effected by selection of mutants (spontaneous orinduced by chemicals or radiation) and/or by recombinant DNA technologye.g. by overexpression or inactivation, respectively, of genes encodingthe enzymes or factors regulating these genes.

In a more preferred host cell, the genetic modification comprisesoverexpression of at least one enzyme of the (non-oxidative part)pentose phosphate pathway. Preferably the enzyme is selected from thegroup consisting of the enzymes encoding for ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, transketolase andtransaldolase, as described in WO 06/009434.

Various combinations of enzymes of the (non-oxidative part) pentosephosphate pathway may be overexpressed. E.g. the enzymes that areoverexpressed may be at least the enzymes ribulose-5-phosphate isomeraseand ribulose-5-phosphate epimerase; or at least the enzymesribulose-5-phosphate isomerase and transketolase; or at least theenzymes ribulose-5-phosphate isomerase and transaldolase; or at leastthe enzymes ribulose-5-phosphate epimerase and transketolase; or atleast the enzymes ribulose-5-phosphate epimerase and transaldolase; orat least the enzymes transketolase and transaldolase; or at least theenzymes ribulose-5-phosphate epimerase, transketolase and transaldolase;or at least the enzymes ribulose-5-phosphate isomerase, transketolaseand transaldolase; or at least the enzymes ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, and transaldolase; or atleast the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, and transketolase. In one embodiment of the invention each ofthe enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase are overexpressed in the hostcell. More preferred is a host cell in which the genetic modificationcomprises at least overexpression of both the enzymes transketolase andtransaldolase as such a host cell is already capable of anaerobic growthon xylose. In fact, under some conditions we have found that host cellsoverexpressing only the transketolase and the transaldolase already havethe same anaerobic growth rate on xylose as do host cells thatoverexpress all four of the enzymes, i.e. the ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, transketolase andtransaldolase. Moreover, host cells overexpressing both of the enzymesribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase arepreferred over host cells overexpressing only the isomerase or only theepimerase as overexpression of only one of these enzymes may producemetabolic imbalances.

There are various means available in the art for overexpression ofenzymes in the cells of the invention. In particular, an enzyme may beoverexpressed by increasing the copy number of the gene coding for theenzyme in the host cell, e.g. by integrating additional copies of thegene in the host cell's genome, by expressing the gene from an episomalmulticopy expression vector or by introducing a episomal expressionvector that comprises multiple copies of the gene.

Alternatively overexpression of enzymes in the host cells of theinvention may be achieved by using a promoter that is not native to thesequence coding for the enzyme to be overexpressed, i.e. a promoter thatis heterologous to the coding sequence to which it is operably linked.Suitable promoters to this end have already been defined herein.

The coding sequence used for overexpression of the enzymes preferably ishomologous to the host cell of the invention. However, coding sequencesthat are heterologous to the host cell of the invention may likewise beapplied, as mentioned in WO 06/009434.

A nucleotide sequence used for overexpression of ribulose-5-phosphateisomerase in the host cell of the invention is a nucleotide sequenceencoding a polypeptide with ribulose-5-phosphate isomerase activity,whereby preferably the polypeptide has an amino acid sequence having atleast 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 17 or wherebythe nucleotide sequence is capable of hybridising with the nucleotidesequence of SEQ ID NO. 18, under moderate conditions, preferably understringent conditions.

A nucleotide sequence used for overexpression of ribulose-5-phosphateepimerase in the host cell of the invention is a nucleotide sequenceencoding a polypeptide with ribulose-5-phosphate epimerase activity,whereby preferably the polypeptide has an amino acid sequence having atleast 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 19 or wherebythe nucleotide sequence is capable of hybridising with the nucleotidesequence of SEQ ID NO. 20, under moderate conditions, preferably understringent conditions.

A nucleotide sequence used for overexpression of transketolase in thehost cell of the invention is a nucleotide sequence encoding apolypeptide with transketolase activity, whereby preferably thepolypeptide has an amino acid sequence having at least 50, 60, 70, 80,90 or 95% identity with SEQ ID NO. 21 or whereby the nucleotide sequenceis capable of hybridising with the nucleotide sequence of SEQ ID NO. 22,under moderate conditions, preferably under stringent conditions.

A nucleotide sequence used for overexpression of transaldolase in thehost cell of the invention is a nucleotide sequence encoding apolypeptide with transaldolase activity, whereby preferably thepolypeptide has an amino acid sequence having at least 50, 60, 70, 80,90 or 95% identity with SEQ ID NO. 23 or whereby the nucleotide sequenceis capable of hybridising with the nucleotide sequence of SEQ ID NO. 24,under moderate conditions, preferably under stringent conditions.

Overexpression of an enzyme, when referring to the production of theenzyme in a genetically modified host cell, means that the enzyme isproduced at a higher level of specific enzymatic activity as compared tothe unmodified host cell under identical conditions. Usually this meansthat the enzymatically active protein (or proteins in case ofmulti-subunit enzymes) is produced in greater amounts, or rather at ahigher steady state level as compared to the unmodified host cell underidentical conditions. Similarly this usually means that the mRNA codingfor the enzymatically active protein is produced in greater amounts, oragain rather at a higher steady state level as compared to theunmodified host cell under identical conditions. Overexpression of anenzyme is thus preferably determined by measuring the level of theenzyme's specific activity in the host cell using appropriate enzymeassays as described herein. Alternatively, overexpression of the enzymemay determined indirectly by quantifying the specific steady state levelof enzyme protein, e.g. using antibodies specific for the enzyme, or byquantifying the specific steady level of the mRNA coding for the enzyme.The latter may particularly be suitable for enzymes of the pentosephosphate pathway for which enzymatic assays are not easily feasible assubstrates for the enzymes are not commercially available. Preferably inthe host cells of the invention, an enzyme to be overexpressed isoverexpressed by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 ascompared to a strain which is genetically identical except for thegenetic modification causing the overexpression. It is to be understoodthat these levels of overexpression may apply to the steady state levelof the enzyme's activity, the steady state level of the enzyme's proteinas well as to the steady state level of the transcript coding for theenzyme.

In a further preferred embodiment, the host cell of the invention:

-   -   expressing araA, araB and araD, and exhibiting the ability to        directly isomerise xylose into xylulose, and optionally    -   comprising a genetic modification that increase the flux of the        pentose pathway as earlier defined herein        further comprises a genetic modification that increases the        specific xylulose kinase activity. Preferably the genetic        modification causes overexpression of a xylulose kinase, e.g. by        overexpression of a nucleotide sequence encoding a xylulose        kinase. The gene encoding the xylulose kinase may be endogenous        to the host cell or may be a xylulose kinase that is        heterologous to the host cell. A nucleotide sequence used for        overexpression of xylulose kinase in the host cell of the        invention is a nucleotide sequence encoding a polypeptide with        xylulose kinase activity, whereby preferably the polypeptide has        an amino acid sequence having at least 50, 60, 70, 80, 90 or 95%        identity with SEQ ID NO. 25 or whereby the nucleotide sequence        is capable of hybridising with the nucleotide sequence of SEQ ID        NO. 26, under moderate conditions, preferably under stringent        conditions.

A particularly preferred xylulose kinase is a xylose kinase that isrelated to the xylulose kinase xylB from Piromyces as mentioned in WO03/0624430. A more preferred nucleotide sequence for use inoverexpression of xylulose kinase in the host cell of the invention is anucleotide sequence encoding a polypeptide with xylulose kinaseactivity, whereby preferably the polypeptide has an amino acid sequencehaving at least 45, 50, 55, 60, 65, 70, 80, 90 or 95% identity with SEQID NO. 27 or whereby the nucleotide sequence is capable of hybridisingwith the nucleotide sequence of SEQ ID NO. 28, under moderateconditions, preferably under stringent conditions.

In the host cells of the invention, genetic modification that increasesthe specific xylulose kinase activity may be combined with any of themodifications increasing the flux of the pentose phosphate pathway asdescribed above, but this combination is not essential for theinvention. Thus, a host cell of the invention comprising a geneticmodification that increases the specific xylulose kinase activity inaddition to the expression of the araA, araB and araD enzymes as definedherein is specifically included in the invention. The various meansavailable in the art for achieving and analysing overexpression of axylulose kinase in the host cells of the invention are the same asdescribed above for enzymes of the pentose phosphate pathway. Preferablyin the host cells of the invention, a xylulose kinase to beoverexpressed is overexpressed by at least a factor 1.1, 1.2, 1.5, 2, 5,10 or 20 as compared to a strain which is genetically identical exceptfor the genetic modification causing the overexpression. It is to beunderstood that these levels of overexpression may apply to the steadystate level of the enzyme's activity, the steady state level of theenzyme's protein as well as to the steady state level of the transcriptcoding for the enzyme.

In a further preferred embodiment, the host cell of the invention:

-   -   expressing araA, araB and araD, and exhibiting the ability to        directly isomerise xylose into xylulose, and optionally    -   comprising a genetic modification that increase the flux of the        pentose pathway and/or    -   further comprising a genetic modification that increases the        specific xylulose kinase activity all as earlier defined herein        further comprises a genetic modification that reduces unspecific        aldose reductase activity in the host cell. Preferably,        unspecific aldose reductase activity is reduced in the host cell        by one or more genetic modifications that reduce the expression        of or inactivate a gene encoding an unspecific aldose reductase,        as described in WO 06/009434. Preferably, the genetic        modifications reduce or inactivate the expression of each        endogenous copy of a gene encoding an unspecific aldose        reductase in the host cell. Host cells may comprise multiple        copies of genes encoding unspecific aldose reductases as a        result of di-, poly- or aneu-ploidy, and/or the host cell may        contain several different (iso)enzymes with aldose reductase        activity that differ in amino acid sequence and that are each        encoded by a different gene. Also in such instances preferably        the expression of each gene that encodes an unspecific aldose        reductase is reduced or inactivated. Preferably, the gene is        inactivated by deletion of at least part of the gene or by        disruption of the gene, whereby in this context the term gene        also includes any non-coding sequence up- or down-stream of the        coding sequence, the (partial) deletion or inactivation of which        results in a reduction of expression of unspecific aldose        reductase activity in the host cell. A nucleotide sequence        encoding an aldose reductase whose activity is to be reduced in        the host cell of the invention is a nucleotide sequence encoding        a polypeptide with aldose reductase activity, whereby preferably        the polypeptide has an amino acid sequence having at least 50,        60, 70, 80, 90 or 95% identity with SEQ ID NO. 29 or whereby the        nucleotide sequence is capable of hybridising with the        nucleotide sequence of SEQ ID NO. 30 under moderate conditions,        preferably under stringent conditions.

In the host cells of the invention, the expression of the araA, araB andaraD enzymes as defined herein is combined with genetic modificationthat reduces unspecific aldose reductase activity. The geneticmodification leading to the reduction of unspecific aldose reductaseactivity may be combined with any of the modifications increasing theflux of the pentose phosphate pathway and/or with any of themodifications increasing the specific xylulose kinase activity in thehost cells as described above, but these combinations are not essentialfor the invention. Thus, a host cell expressing araA, araB, and araD,comprising an additional genetic modification that reduces unspecificaldose reductase activity is specifically included in the invention.

In a preferred embodiment, the host cell is CBS120327 deposited at theCBS Institute (The Netherlands) on Sep. 27, 2006.

In a further preferred embodiment, the invention relates to modifiedhost cells that are further adapted to L-arabinose (use L-arabinoseand/or convert it into L-ribulose, and/or xylulose 5-phosphate and/orinto a desired fermentation product and optionally xylose utilisation byselection of mutants, either spontaneous or induced (e.g. by radiationor chemicals), for growth on L-arabinose and optionally xylose,preferably on L-arabinose and optionally xylose as sole carbon source,and more preferably under anaerobic conditions. Selection of mutants maybe performed by serial passaging of cultures as e.g. described by Kuyperet al. (2004, FEMS Yeast Res. 4: 655-664) and/or by cultivation underselective pressure in a chemostat culture as is described in Example 4of WO 06/009434. This selection process may be continued as long asnecessary. This selection process is preferably carried out during oneweek till one year. However, the selection process may be carried outfor a longer period of time if necessary. During the selection process,the cells are preferably cultured in the presence of approximately 20g/l L-arabinose and/or approximately 20 g/l xylose. The cell obtained atthe end of this selection process is expected to be improved as to itscapacities of using L-arabinose and/or xylose, and/or convertingL-arabinose into L-ribulose and/or xylulose 5-phosphate and/or a desiredfermentation product such as ethanol. In this context “improved cell”may mean that the obtained cell is able to use L-arabinose and/or xylosein a more efficient way than the cell it derives from. For example, theobtained cell is expected to better grow: increase of the specificgrowth rate of at least 2% than the cell it derives from under the sameconditions. Preferably, the increase is of at least 4%, 6%, 8%, 10%,15%, 20%, 25% or more. The specific growth rate may be calculated fromOD₆₆₀ as known to the skilled person. Therefore, by monitoring theOD₆₆₀, one can deduce the specific growth rate. In this context“improved cell” may also mean that the obtained cell convertsL-arabinose into L-ribulose and/or xylulose 5-phosphate and/or a desiredfermentation product such as ethanol in a more efficient way than thecell it derives from. For example, the obtained cell is expected toproduce higher amounts of L-ribulose and/or xylulose 5-phosphate and/ora desired fermentation product such as ethanol: increase of at least oneof these compounds of at least 2% than the cell it derives from underthe same conditions. Preferably, the increase is of at least 4%, 6%, 8%,10%, 15%, 20%, 25% or more. In this context “improved cell” may alsomean that the obtained cell converts xylose into xylulose and/or adesired fermentation product such as ethanol in a more efficient waythan the cell it derives from. For example, the obtained cell isexpected to produce higher amounts of xylulose and/or a desiredfermentation product such as ethanol: increase of at least one of thesecompounds of at least 2% than the cell it derives from under the sameconditions. Preferably, the increase is of at least 4%, 6%, 8%, 10%,15%, 20%, 25% or more.

In a preferred host cell of the invention at least one of the geneticmodifications described above, including modifications obtained byselection of mutants, confer to the host cell the ability to grow onL-arabinose and optionally xylose as carbon source, preferably as solecarbon source, and preferably under anaerobic conditions. Preferably themodified host cell produce essentially no xylitol, e.g. the xylitolproduced is below the detection limit or e.g. less than 5, 2, 1, 0.5, or0.3% of the carbon consumed on a molar basis.

Preferably the modified host cell has the ability to grow on L-arabinoseand optionally xylose as sole carbon source at a rate of at least 0.001,0.005, 0.01, 0.03, 0.05, 0.1, 0.2, 0.25 or 0.3 h⁻¹ under aerobicconditions, or, if applicable, at a rate of at least 0.001, 0.005, 0.01,0.03, 0.05, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15 or 0.2 h⁻¹ under anaerobicconditions Preferably the modified host cell has the ability to grow ona mixture of glucose and L-arabinose and optionally xylose (in a 1:1weight ratio) as sole carbon source at a rate of at least 0.001, 0.005,0.01, 0.03, 0.05, 0.1, 0.2, 0.25 or 0.3 h⁻¹ under aerobic conditions,or, if applicable, at a rate of at least 0.001, 0.005, 0.01, 0.03, 0.05,0.1, 0.12, 0.15, or 0.2 h⁻¹ under anaerobic conditions.

Preferably, the modified host cell has a specific L-arabinose andoptionally xylose consumption rate of at least 346, 350, 400, 500, 600,650, 700, 750, 800, 900 or 1000 mg/g cells/h. Preferably, the modifiedhost cell has a yield of fermentation product (such as ethanol) onL-arabinose and optionally xylose that is at least 20, 25, 30, 35, 40,45, 50, 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell's yield offermentation product (such as ethanol) on glucose. More preferably, themodified host cell's yield of fermentation product (such as ethanol) onL-arabinose and optionally xylose is equal to the host cell's yield offermentation product (such as ethanol) on glucose. Likewise, themodified host cell's biomass yield on L-arabinose and optionally xyloseis preferably at least 55, 60, 70, 80, 85, 90, 95 or 98% of the hostcell's biomass yield on glucose. More preferably, the modified hostcell's biomass yield on L-arabinose and optionally xylose is equal tothe host cell's biomass yield on glucose. It is understood that in thecomparison of yields on glucose and L-arabinose and optionally xyloseboth yields are compared under aerobic conditions or both underanaerobic conditions.

In a more preferred embodiment, the host cell is CBS120328 deposited atthe CBS Institute (The Netherlands) on Sep. 27, 2006 or CBS121879deposited at the CBS Institute (The Netherlands) on Sep. 20, 2007.In a preferred embodiment, the cell expresses one or more enzymes thatconfer to the cell the ability to produce at least one fermentationproduct selected from the group consisting of ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin.In a more preferred embodiment, the host cell of the invention is a hostcell for the production of ethanol. In another preferred embodiment, theinvention relates to a transformed host cell for the production offermentation products other than ethanol. Such non-ethanolicfermentation products include in principle any bulk or fine chemicalthat is producible by a eukaryotic microorganism such as a yeast or afilamentous fungus. Such fermentation products include e.g. lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin.A preferred host cell of the invention for production of non-ethanolicfermentation products is a host cell that contains a geneticmodification that results in decreased alcohol dehydrogenase activity.

Method

In a further aspect, the invention relates to fermentation processes inwhich a host cell of the invention is used for the fermentation of acarbon source comprising a source of L-arabinose and optionally a sourceof xylose. Preferably, the source of L-arabinose and the source ofxylose are L-arabinose and xylose. In addition, the carbon source in thefermentation medium may also comprise a source of glucose. The source ofL-arabinose, xylose or glucose may be L-arabinose, xylose or glucose assuch or may be any carbohydrate oligo- or polymer comprisingL-arabinose, xylose or glucose units, such as e.g. lignocellulose,xylans, cellulose, starch, arabinan and the like. For release of xyloseor glucose units from such carbohydrates, appropriate carbohydrases(such as xylanases, glucanases, amylases and the like) may be added tothe fermentation medium or may be produced by the modified host cell. Inthe latter case the modified host cell may be genetically engineered toproduce and excrete such carbohydrases. An additional advantage of usingoligo- or polymeric sources of glucose is that it enables to maintain alow(er) concentration of free glucose during the fermentation, e.g. byusing rate-limiting amounts of the carbohydrases. This, in turn, willprevent repression of systems required for metabolism and transport ofnon-glucose sugars such as xylose. In a preferred process the modifiedhost cell ferments both the L-arabinose (optionally xylose) and glucose,preferably simultaneously in which case preferably a modified host cellis used which is insensitive to glucose repression to prevent diauxicgrowth. In addition to a source of L-arabinose, optionally xylose (andglucose) as carbon source, the fermentation medium will further comprisethe appropriate ingredient required for growth of the modified hostcell. Compositions of fermentation media for growth of microorganismssuch as yeasts or filamentous fungi are well known in the art.

In a preferred process, there is provided a process for producing afermentation product selected from the group consisting of ethanol,lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid,succinic acid, citric acid, malic acid, fumaric acid, an amino acid,1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic anda cephalosporin whereby the process comprises the steps of:

-   (a) fermenting a medium containing a source of L-arabinose and    optionally xylose with a modified host cell as defined herein,    whereby the host cell ferments L-arabinose and optionally xylose to    the fermentation product, and optionally,-   (b) recovering the fermentation product.    The fermentation process is a process for the production of a    fermentation product such as e.g. ethanol, lactic acid,    3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,    citric acid, malic acid, fumaric acid, an amino acid,    1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam    antibiotic, such as Penicillin G or Penicillin V and fermentative    derivatives thereof, and/or a cephalosporin. The fermentation    process may be an aerobic or an anaerobic fermentation process. An    anaerobic fermentation process is herein defined as a fermentation    process run in the absence of oxygen or in which substantially no    oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more    preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not    detectable), and wherein organic molecules serve as both electron    donor and electron acceptors. In the absence of oxygen, NADH    produced in glycolysis and biomass formation, cannot be oxidised by    oxidative phosphorylation. To solve this problem many microorganisms    use pyruvate or one of its derivatives as an electron and hydrogen    acceptor thereby regenerating NAD⁺. Thus, in a preferred anaerobic    fermentation process pyruvate is used as an electron (and hydrogen    acceptor) and is reduced to fermentation products such as ethanol,    lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid,    succinic acid, citric acid, malic acid, fumaric acid, an amino acid,    1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam    antibiotics and a cephalosporin. In a preferred embodiment, the    fermentation process is anaerobic. An anaerobic process is    advantageous since it is cheaper than aerobic processes: less    special equipment is needed. Furthermore, anaerobic processes are    expected to give a higher product yield than aerobic processes.    Under aerobic conditions, usually the biomass yield is higher than    under anaerobic conditions. As a consequence, usually under aerobic    conditions, the expected product yield is lower than under anaerobic    conditions. According to the inventors, the process of the invention    is the first anaerobic fermentation process with a medium comprising    a source of L-arabinose that has been developed so far.    In another preferred embodiment, the fermentation process is under    oxygen-limited conditions. More preferably, the fermentation process    is aerobic and under oxygen-limited conditions. An oxygen-limited    fermentation process is a process in which the oxygen consumption is    limited by the oxygen transfer from the gas to the liquid. The    degree of oxygen limitation is determined by the amount and    composition of the ingoing gasflow as well as the actual mixing/mass    transfer properties of the fermentation equipment used. Preferably,    in a process under oxygen-limited conditions, the rate of oxygen    consumption is at least 5.5, more preferably at least 6 and even    more preferably at least 7 mmol/L/h.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably less than 38° C. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C.

A preferred process is a process for the production of ethanol, wherebythe process comprises the steps of: (a) fermenting a medium containing asource of L-arabinose and optionally xylose with a modified host cell asdefined herein, whereby the host cell ferments L-arabinose andoptionally xylose to ethanol; and optionally, (b) recovery of theethanol. The fermentation medium may also comprise a source of glucosethat is also fermented to ethanol. In a preferred embodiment, thefermentation process for the production of ethanol is anaerobic.Anaerobic has already been defined earlier herein. In another preferredembodiment, the fermentation process for the production of ethanol isaerobic. In another preferred embodiment, the fermentation process forthe production of ethanol is under oxygen-limited conditions, morepreferably aerobic and under oxygen-limited conditions. Oxygen-limitedconditions have already been defined earlier herein.

In the process, the volumetric ethanol productivity is preferably atleast 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre perhour. The ethanol yield on L-arabinose and optionally xylose and/orglucose in the process preferably is at least 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as apercentage of the theoretical maximum yield, which, for glucose andL-arabinose and optionally xylose is 0.51 g. ethanol per g. glucose orxylose. In another preferred embodiment, the invention relates to aprocess for producing a fermentation product selected from the groupconsisting of lactic acid, 3-hydroxy-propionic acid, acrylic acid,acetic acid, succinic acid, citric acid, malic acid, fumaric acid, anamino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactamantibiotic and a cephalosporin. The process preferably comprises thesteps of (a) fermenting a medium containing a source of L-arabinose andoptionally xylose with a modified host cell as defined herein above,whereby the host cell ferments L-arabinose and optionally xylose to thefermentation product, and optionally, (b) recovery of the fermentationproduct. In a preferred process, the medium also contains a source ofglucose.

In the fermentation process of the invention leading to the productionof ethanol, several advantages can be cited by comparison to knownethanol fermentations processes:

-   -   anaerobic processes are possible.    -   oxygen limited conditions are also possible.    -   higher ethanol yields and ethanol production rates can be        obtained.    -   the strain used may be able to use L-arabinose and optionally        xylose.        Alternatively to the fermentation processes described above,        another fermentation process is provided as a further aspect of        the invention wherein, at least two distinct cells are used for        the fermentation of a carbon source comprising at least two        sources of carbon selected from the group consisting of but not        limited thereto: a source of L-arabinose, a source of xylose and        a source of glucose. In this fermentation process, “at least two        distinct cells” means this process is preferably a        co-fermentation process. In one preferred embodiment, two        distinct cells are used: one being the one of the invention as        earlier defined able to use L-arabinose, and/or to convert it        into L-ribulose, and/or xylulose 5-phosphate and/or into a        desired fermentation product such as ethanol and optionally        being able to use xylose, the other one being for example a        strain which is able to use xylose and/or convert it into a        desired fermentation product such as ethanol as defined in WO        03/062430 and/or WO 06/009434. A cell which is able to use        xylose is preferably a strain which exhibits the ability of        directly isomerising xylose into xylulose (in one step) as        earlier defined herein. These two distinct strains are        preferably cultivated in the presence of a source of        L-arabinose, a source of xylose and optionally a source of        glucose. Three distinct cells or more may be co-cultivated        and/or three or more sources of carbon may be used, provided at        least one cell is able to use at least one source of carbon        present and/or to convert it into a desired fermentation product        such as ethanol. The expression “use at least one source of        carbon” has the same meaning as the expression “use of        L-arabinose”. The expression “convert it (i.e. a source of        carbon) into a desired fermentation product has the same meaning        as the expression “convert L-arabinose into a desired        fermentation product”.        In a preferred embodiment, the invention relates to a process        for producing a fermentation product selected from the group        consisting of ethanol, lactic acid, 3-hydroxy-propionic acid,        acrylic acid, acetic acid, succinic acid, citric acid, malic        acid, fumaric acid, amino acids, 1,3-propane-diol, ethylene,        glycerol, butanol, β-lactam antibiotics and cephalosporins,        whereby the process comprises the steps of:    -   (a) fermenting a medium containing at least a source of        L-arabinose and a source of xylose with a cell of the invention        as earlier defined herein and a cell able to use xylose and/or        exhibiting the ability to directly isomerise xylose into        xylulose, whereby each cell ferments L-arabinose and/or xylose        to the fermentation product, and optionally,    -   (b) recovering the fermentation product.        All preferred embodiments of the fermentation processes as        described above are also preferred embodiments of this further        fermentation processes: identity of the fermentation product,        identity of source of L-arabinose and source of xylose,        conditions of fermentation (aerobical or anaerobical conditions,        oxygen-limited conditions, temperature at which the process is        being carried out, productivity of ethanol, yield of ethanol).

Genetic Modifications

For overexpression of enzymes in the host cells of the inventions asdescribed above, as well as for additional genetic modification of hostcells, preferably yeasts, host cells are transformed with the variousnucleic acid constructs of the invention by methods well known in theart. Such methods are e.g. known from standard handbooks, such asSambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, or F. Ausubel et al, eds., “Current protocols in molecularbiology”, Green Publishing and Wiley Interscience, New York (1987).Methods for transformation and genetic modification of fungal host cellsare known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO00/37671.

Promoters for use in the nucleic acid constructs for overexpression ofenzymes in the host cells of the invention have been described above. Inthe nucleic acid constructs for overexpression, the 3′-end of thenucleotide acid sequence encoding the enzyme(s) preferably is operablylinked to a transcription terminator sequence. Preferably the terminatorsequence is operable in a host cell of choice, such as e.g. the yeastspecies of choice. In any case the choice of the terminator is notcritical; it may e.g. be from any yeast gene, although terminators maysometimes work if from a non-yeast, eukaryotic, gene. The transcriptiontermination sequence further preferably comprises a polyadenylationsignal. Preferred terminator sequences are the alcohol dehydrogenase(ADH1) and the PGI1 terminators. More preferably, the ADH1 and the PGI1terminators are both from S. cerevisiae (SEQ ID NO:50 and SEQ ID NO:53respectively).

Optionally, a selectable marker may be present in the nucleic acidconstruct. As used herein, the term “marker” refers to a gene encoding atrait or a phenotype which permits the selection of, or the screeningfor, a host cell containing the marker. The marker gene may be anantibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among cells that are nottransformed. Preferably however, non-antibiotic resistance markers areused, such as auxotrophic markers (URA3, TRP1, LEU2). In a preferredembodiment the host cells transformed with the nucleic acid constructsare marker gene free. Methods for constructing recombinant marker genefree microbial host cells are disclosed in EP-A-0 635 574 and are basedon the use of bidirectional markers. Alternatively, a screenable markersuch as Green Fluorescent Protein, lacZ, luciferase, chloramphenicolacetyltransferase, beta-glucuronidase may be incorporated into thenucleic acid constructs of the invention allowing to screen fortransformed cells.

Optional further elements that may be present in the nucleic acidconstructs of the invention include, but are not limited to, one or moreleader sequences, enhancers, integration factors, and/or reporter genes,intron sequences, centromers, telomers and/or matrix attachment (MAR)sequences. The nucleic acid constructs of the invention may furthercomprise a sequence for autonomous replication, such as an ARS sequence.Suitable episomal nucleic acid constructs may e.g. be based on the yeast2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids.Alternatively the nucleic acid construct may comprise sequences forintegration, preferably by homologous recombination. Such sequences maythus be sequences homologous to the target site for integration in thehost cell's genome. The nucleic acid constructs of the invention can beprovided in a manner known per se, which generally involves techniquessuch as restricting and linking nucleic acids/nucleic acid sequences,for which reference is made to the standard handbooks, such as Sambrookand Russel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd)edition), Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress.

Methods for inactivation and gene disruption in yeast or fungi are wellknown in the art (see e.g. Fincham, 1989, Microbiol Rev. 53(1):148-70and EP-A-0 635 574).

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

The invention is further described by the following examples, whichshould not be construed as limiting the scope of the invention.

EXAMPLES Plasmid and Strain Construction Strains

The L-arabinose consuming Sachharomyces cerevisiae strain described inthis work is based on strain RWB220, which is itself a derivative ofRWB217. RWB217 is a CEN.PK strain in which four genes coding for theexpression of enzymes in the pentose phosphate pathway have beenoverexpressed, TAL1, TKL1, RPE1, RKI1 (Kuyper et al., 2005a). Inaddition the gene coding for an aldose reductase (GRE3), has beendeleted. Strain RWB217 also contains two plasmids, a single copy plasmidwith a LEU2 marker for overexpression of the xylulokinase (XKS1) and anepisomal, multicopy plasmid with URA3 as the marker for the expressionof the xylose isomerase, XylA. RWB217 was subjected to a selectionprocedure for improved growth on xylose which is described in Kuyper etal. (2005b). The procedure resulted in two pure strains, RWB218 (Kuyperet al., 2005b) and RWB219. The difference between RWB218 and RWB219 isthat after the selection procedure, RWB218 was obtained by plating andrestreaking on mineral medium with glucose as the carbon source, whilefor RWB219 xylose was used.

Strain RWB219 was grown non-selectively on YP with glucose (YPD) as thecarbon source in order to facilitate the loss of both plasmids. Afterplating on YPD single colonies were tested for plasmid loss by lookingat uracil and leucine auxotrophy. A strain that had lost both plasmidswas transformed with pSH47, containing the cre recombinase, in order toremove a KanMX cassette (Guldener et al., 1996), still present afterintegrating the RKIJ overexpression construct. Colonies with the plasmidwere resuspended in Yeast Peptone medium (YP) (10 g/l yeast extract and20 g/l peptone both from BD Difco Belgium) with 1% galactose andincubated for 1 hour at 30° C. About 200 cells were plated on YPD. Theresulting colonies were checked for loss of the KanMX marker (G418resistance) and pSH47 (URA3). A strain that had lost both the KanMXmarker and the pSH47 plasmid was then named RWB220. To obtain the straintested in this patent, RWB220 was transformed with pRW231 and pRW243(table 2), resulting in strain IMS0001.

During construction strains were maintained on complex YP: 10 gl⁻¹ yeastextract (BD Difco), 20 gl⁻¹ peptone (BD Difco) or synthetic medium (MY)(Verduyn et al., 1992) supplemented with glucose (2%) as carbon source(YPD or MYD) and 1.5% agar in the case of plates. After transformationwith plasmids strains were plated on MYD. Transformations of yeast weredone according to Gietz and Woods (2002). Plasmids were amplified inEscherichia coli strain XL-1 blue (Stratagene, La Jolla, Calif., USA).Transformation was performed according to Inoue et al. (1990). E. coliwas grown on LB (Luria-Bertani) plates or in liquid TB (Terrific Broth)medium for the isolation of plasmids (Sambrook et al, 1989).

Plasmids

In order to grow on L-arabinose, yeast needs to express three differentgenes, an L-arabinose isomerase (AraA), a L-ribulokinase (AraB), and aL-ribulose-5-P 4-epimerase (AraD) (Becker and Boles, 2003). In this workwe have chosen to express AraA, AraB, and AraD from the lactic acidbacterium Lactobacillus plantarum in S. cerevisiae. Because the eventualaim is to consume L-arabinose in combination with other sugars, likeD-xylose, the genes encoding the bacterial L-arabinose pathway werecombined on the same plasmid with the genes coding for D-xyloseconsumption.

In order to get a high level of expression, the L. plantarum AraA andAraD genes were ligated into plasmid pAKX002, the 2μ XylA bearingplasmid.

The AraA cassette was constructed by amplifying a truncated version ofthe TDH3 promoter with SpeIS'Ptdh3 and 5′AraAPtdh3 (SEQ ID NO: 49), theAraA gene with Ptdh5′AraA and Tadh3′AraA and the ADH1 terminator (SEQ IDNO:50) with 3′AraATadhl and 3′Tadhl-SpeI. The three fragments wereextracted from gel and mixed in roughly equimolar amounts. On thismixture a PCR was performed using the SpeI-5′Ptdh3 and 3′TadhlSpeIoligos. The resulting P_(TDH3)-AraA-T_(ADH1) cassette was gel purified,cut at the 5′ and 3′ SpeI sites and then ligated into pAKX002 cut withNheI, resulting in plasmid pRW230.

The AraD construct was made by first amplifying a truncated version ofthe HXT7 promoter (SEQ ID NO:52) with oligos SalIS'Phxt7 and 5′AraDPhxt,the AraD gene with Phxt5′AraD and Tpgi3′AraD and the GPI1 terminator(SEQ ID NO:53) region with the 3′AraDTpgi and 3′TpgiSalI oligos. Theresulting fragments were extracted from gel and mixed in roughlyequimolar amounts, after which a PCR was performed using the SalIS'Phxt7and 3′TpgilSalI oligos. The resulting P_(HXT7)-AraD-T_(PGI1) cassettewas gel purified, cut at the 5′ and 3′ SalI sites and then ligated intopRW230 cut with XhoI, resulting in plasmid pRW231 (FIG. 1).

Since too high an expression of the L-ribulokinase is detrimental togrowth (Becker and Boles, 2003), the AraB gene was combined with theXKS1 gene, coding for xylulokinase, on an integration plasmid. For this,p415ADHXKS (Kuyper et al., 2005a) was first changed into pRW229, bycutting both p415ADHXKS and pRS305 with PvuI and ligating theADHXKS-containing PvuI fragment from p415ADHXKS to the vector backbonefrom pRS305, resulting in pRW229.

A cassette, containing the L. plantarum AraB gene between the PGI1promoter (SEQ ID NO:51) and ADH1 terminator (SEQ ID NO:50) was made byamplifying the PGI1 promoter with the SacIS'Ppgi1 and 5′AraBPpgi1oligos, the AraB gene with the Ppgi5′AraB and Tadh3′AraB oligos and theADH1 terminator with 3′AraBTadh1 and 3′TadhlSacI oligos. The threefragments were extracted from gel and mixed in roughly equimolaramounts. On this mixture a PCR was performed using the SacI-5′Ppgi1 and3′TadhlSacI oligos. The resulting P_(PGI1)-AraB-T_(ADH1) cassette wasgel purified, cut at the 5′ and 3′ Sad sites and then ligated intopRW229 cut with Sad, resulting in plasmid pRW243 (FIG. 1).

Strain RWB220 was transformed with pRW231 and pRW243 (table 2),resulting in strain IMS0001.

Restriction endonucleases (New England Biolabs, Beverly, Mass., USA andRoche, Basel, Switzerland) and DNA ligase (Roche) were used according tothe manufacturers' specifications. Plasmid isolation from E. coli wasperformed with the Qiaprep spin miniprep kit (Qiagen, Hilden, Germany).DNA fragments were separated on a 1% agarose (Sigma, St. Louis, Mo.,USA) gel in 1×TBE (Sambrook et al, 1989). Isolation of fragments fromgel was carried out with the Qiaquick gel extraction kit (Quiagen).Amplification of the (elements of the) AraA, AraB and AraD cassettes wasdone with Vent_(R) DNA polymerase (New England Biolabs) according to themanufacturer's specification. The template was chromosomal DNA of S.cerevisiae CEN.PK113-7D for the promoters and terminators, orLactobacillus plantarum DSM20205 for the Ara genes. The polymerase chainreaction (PCR) was performed in a Biometra TGradient Thermocycler(Biometra, Gottingen, Germany) with the following settings: 30 cycles of1 min annealing at 55° C., 60° C. or 65° C., 1 to 3 min extension at 75°C., depending on expected fragment size, and 1 min denaturing at 94° C.

Cultivation and Media

Shake-flask cultivations were performed at 30° C. in a synthetic medium(Verduyn et al., 1992). The pH of the medium was adjusted to 6.0 with 2M KOH prior to sterilisation. For solid synthetic medium, 1.5% of agarwas added.

Pre-cultures were prepared by inoculating 100 ml medium containing theappropriate sugar in a 500-ml shake flask with a frozen stock culture.After incubation at 30° C. in an orbital shaker (200 rpm), this culturewas used to inoculate either shake-flask cultures or fermenter cultures.The synthetic medium for anaerobic cultivation was supplemented with0.01 gl⁻¹ ergosterol and 0.42 gl⁻¹ Tween 80 dissolved in ethanol(Andreasen and Stier, 1953; Andreasen and Stier, 1954). Anaerobic(sequencing) batch cultivation was carried out at 30° C. in 2-1laboratory fermenters (Applikon, Schiedam, The Netherlands) with aworking volume of 1 l. The culture pH was maintained at pH 5.0 byautomatic addition of 2 M KOH. Cultures were stirred at 800 rpm andsparged with 0.5 l min⁻¹ nitrogen gas (<10 ppm oxygen). To minimisediffusion of oxygen, fermenters were equipped with Norprene tubing (ColePalmer Instrument company, Vernon Hills, USA). Dissolved oxygen wasmonitored with an oxygen electrode (Applisens, Schiedam, TheNetherlands). Oxygen-limited conditions were achieved in the sameexperimental set-up by headspace aeration at approximately 0.05 l min⁻¹.

Determination of Dry Weight

Culture samples (10.0 ml) were filtered over preweighed nitrocellulosefilters (pore size 0.45 μm; Gelman laboratory, Ann Arbor, USA). Afterremoval of medium, the filters were washed with demineralised water anddried in a microwave oven (Bosch, Stuttgart, Germany) for 20 min at 360W and weighed. Duplicate determinations varied by less than 1%.

Gas Analysis

Exhaust gas was cooled in a condensor (2° C.) and dried with a Permapuredryer type MD-110-48P-4 (Permapure, Toms River, USA). O2 and CO2concentrations were determined with a NGA 2000 analyser (RosemountAnalytical, Orrville, USA). Exhaust gasflow rate and specificoxygen-consumption and carbondioxide production rates were determined asdescribed previously (Van Urk et al., 1988; Weusthuis et al., 1994). Incalculating these biomass-specific rates, volume changes caused bywithdrawing culture samples were taken account for.

Metabolite Analysis

Glucose, xylose, arabinose, xylitol, organic acids, glycerol and ethanolwere analysed by HPLC using a Waters Alliance 2690 HPLC (Waters,Milford, USA) supplied with a BioRad HPX 87H column (BioRad, Hercules,USA), a Waters 2410 refractive-index detector and a Waters 2487 UVdetector. The column was eluted at 60° C. with 0.5 gl⁻¹ sulphuric acidat a flow rate of 0.6 ml min⁻¹.

Assay for Xylulose 5-Phosphate (Zaldivar J., et al, Appl. Microbiol.Biotechnol., (2002), 59:436-442)

For the analysis of intracellular metabolites such as xylulose5-phosphate, 5 ml broth was harvested in duplicate from the reactors,before glucose exhaustion (at 22 and 26 h of cultivation) and afterglucose exhaustion (42, 79 and 131 h of cultivation). Procedures formetabolic arrest, solid-phase extraction of metabolites and analysishave been described in detail by Smits H. P. et al. (Anal. Biochem.,261:36-42, (1998)). However, the analysis by high-pressure ion exchangechromatography coupled to pulsed amperometric detection used to analyzecell extracts, was slightly modified. Solutions used were eluent A, 75mM NaOH, and eluent B, 500 mM NaAc. To prevent contamination ofcarbonate in the eluent solutions, a 50% NaOH solution with lowcarbonate concentration (Baker Analysed, Deventer, The Netherlands) wasused instead of NaOH pellets. The eluents were degassed with Helium (He)for 30 min and then kept under a He atmosphere. The gradient pump wasprogrammed to generate the following gradients: 100% A and 0% B (0 min),a linear decrease of A to 70% and a linear increase of B to 30% (0-30min), a linear decrease of A to 30% and a linear increase of B to 70%(30-70 min), a linear decrease of A to 0% and a linear increase of B to100% (70-75 min), 0% A and 100% B (75-85 min), a linear increase of A to100% and a linear decrease of B to 0% (85-95 min) The mobile phase wasrun at a flow rate of 1 ml/min. Other conditions were according to Smitset al. (1998).

Carbon Recovery

Carbon recoveries were calculated as carbon in products formed, dividedby the total amount of sugar carbon consumed, and were based on a carboncontent of biomass of 48%. To correct for ethanol evaporation during thefermentations, the amount of ethanol produced was assumed to be equal tothe measured cumulative production of CO₂ minus the CO₂ production thatoccurred due to biomass synthesis (5.85 mmol CO₂ per gram biomass(Verduyn et al., 1990)) and the CO₂ associated with acetate formation.

Selection for Growth on L-Arabinose

Strain IMS0001 (CBS120327 deposited at the CBS on Sep. 27, 2006),containing the genes encoding the pathways for both xylose (Xy1A andXKS1) and arabinose (AraA, AraB, AraD) metabolization, was constructedaccording the procedure described above. Although capable of growing onxylose (data not shown), strain IMS0001 did not seem to be capable ofgrowing on solid synthetic medium supplemented with 2% L-arabinose.Mutants of IMS0001 capable of utilizing L-arabinose as carbon source forgrowth were selected by serial transfer in shake flasks and bysequencing-batch cultivation in fermenters (SBR).

For the serial transfer experiments, a 500-ml shake flask containing 100ml synthetic medium containing 0.5% galactose were inoculated witheither strain IMS0001, or the reference strain RWB219. After 72 hours,at an optical density at 660 nm of 3.0, the cultures were used toinoculate a new shake flask containing 0.1% galactose and 2% arabinose.Based on HPLC determination with D-ribulose as calibration standard, itwas determined that already in the first cultivations of strain IMS0001,on medium containing a galactose/arabinose mixture, part of thearabinose was converted into ribulose and subsequently excreted to thesupernatant. These HPLC analyses were performed using a Waters Alliance2690 HPLC (Waters, Milford, USA) supplied with a BioRad HPX 87H column(BioRad, Hercules, USA), a Waters 2410 refractive-index detector and aWaters 2487 UV detector. The column was eluted at 60° C. with 0.5 gl⁻¹sulphuric acid at a flow rate of 0.6 ml min⁻¹. In contrast to thereference strain RWB219, the OD₆₆₀ of the culture of strain IMS0001increased after depletion of the galactose. When after approximately 850hours growth on arabinose by strain IMS0001 was observed (FIG. 2), thisculture was transferred at an OD₆₆₀ of 1.7 to a shake flask containing2% arabinose. Cultures were then sequentially transferred to freshmedium containing 2% arabinose at an OD₆₆₀ of 2-3. Utilization ofarabinose was confirmed by occasionally measuring arabinoseconcentrations by HPLC (data not shown). The growth rate of thesecultures increased from 0 to 0.15 h⁻¹ in approximately 3600 hours (FIG.3).

A batch fermentation under oxygen limited conditions was started byinoculating 1 l of synthetic medium supplemented with 2% of arabinosewith a 100 ml shake flask culture of arabinose-grown IMS0001 cells witha maximum growth rate on 2% of L-arabinose of approximately 0.12 h⁻¹.When growth on arabinose was observed, the culture was subjected toanaerobic conditions by sparging with nitrogen gas. The sequentialcycles of anaerobic batch cultivation were started by either manual orautomated replacement of 90% of the culture with synthetic medium with20 gl⁻¹ arabinose. For each cycle during the SBR fermentation, theexponential growth rate was estimated from the CO₂ profile (FIG. 4). In13 cycles, the exponential growth rate increased from 0.025 to 0.08 h⁻¹.After 20 cycles a sample was taken, and plated on solid synthetic mediumsupplemented with 2% of L-arabinose and incubated at 30° C. for severaldays. Separate colonies were re-streaked twice on solid synthetic mediumwith L-arabinose. Finally, a shake flask containing synthetic mediumwith 2% of L-L-arabinose was inoculated with a single colony, andincubated for 5 days at 30° C. This culture was designated as strainIMS0002 (CBS120328 deposited at the Centraal Bureau voorSchimmelculturen (CBS) on Sep. 27, 2006). Culture samples were taken,30% of glycerol was added and samples were stored at −80° C.

Mixed Culture Fermentation

Biomass hydrolysates, a desired feedstock for industrial biotechnology,contain complex mixtures consisting of various sugars amongst whichglucose, xylose and arabinose are commonly present in significantfractions. To accomplish ethanolic fermentation of not only glucose andarabinose, but also xylose, an anaerobic batch fermentation wasperformed with a mixed culture of the arabinose-fermenting strainIMS0002, and the xylose-fermenting strain RWB218. An anaerobic batchfermenter containing 800 ml of synthetic medium supplied with 30 gl⁻¹D-glucose, 15 gl⁻¹ D-xylose, and 15 gl⁻¹ L-arabinose was inoculated with100 ml of pre-culture of strain IMS0002. After 10 hours, a 100 mlinoculum of RWB218 was added. In contrast to the mixed sugarfermentation with only strain IMS0002, both xylose and arabinose wereconsumed after glucose depletion (FIG. 5D). The mixed culture completelyconsumed all sugars, and within 80 hours 564.0±6 3 mmol 1⁻¹ ethanol(calculated from the CO₂ production) was produced with a high overallyield of 0.42 g g⁻¹ sugar. Xylitol was produced only in small amounts,to a concentration of 4.7 mmol 1⁻¹.

Characterization of Strain IMS0002

Growth and product formation of strain IMS0002 was determined duringanaerobic batch fermentations on synthetic medium with eitherL-arabinose as the sole carbon source, or a mixture of glucose, xyloseand L-arabinose. The pre-cultures for these anaerobic batchfermentations were prepared in shake flasks containing 100 ml ofsynthetic medium with 2% L-arabinose, by inoculating with −80° C. frozenstocks of strain IMS0002, and incubating for 48 hours at 30° C.

FIG. 5A shows that strain IMS0002 is capable of fermenting 20 gl⁻¹L-arabinose to ethanol during an anaerobic batch fermentation ofapproximately 70 hours. The specific growth rate under anaerobicconditions with L-arabinose as sole carbon source was 0.05±0.001 h⁻¹.Taking into account the ethanol evaporation during the batchfermentation, the ethanol yield from 20 gl⁻¹ arabinose was 0.43±0.003 gg⁻¹. Without evaporation correction the ethanol yield was 0.35±0.01 gg⁻¹ of arabinose. No formation of arabinitol was observed duringanaerobic growth on arabinose. In FIG. 5B, the ethanolic fermentation ofa mixture of 20 gl⁻¹ glucose and 20 g 1⁻¹ L-arabinose by strain IMS0002is shown. L-arabinose consumption started after glucose depletion.Within 70 hours, both the glucose and L-arabinose were completelyconsumed. The ethanol yield from the total of sugars was 0.42±0.003 gg⁻¹.

In FIG. 5C, the fermentation profile of a mixture of 30 gl⁻¹ glucose, 15gl⁻¹ D-xylose, and 15 gl⁻¹ L-arabinose by strain IMS0002 is shown.Arabinose consumption started after glucose depletion. Within 80 hours,both the glucose and arabinose were completely consumed. Only 20 mM from100 mM of xylose was consumed by strain IMS0002. In addition, theformation of 20 mM of xylitol was observed. Apparently, the xylose wasconverted into xylitol by strain IMS0002. Hence, the ethanol yield fromthe total of sugars was lower than for the above describedfermentations: 0.38±0.001 g g⁻¹. The ethanol yield from the total ofglucose and arabinose was similar to the other fermentations:0.43±0.001¹ g g⁻¹.

Table 1 shows the arabinose consumption rates and the ethanol productionrates observed for the anaerobic batch fermentation of strain IMS0002.Arabinose was consumed with a rate of 0.23-0.75 g h⁻¹ g⁻¹ biomass dryweight. The rate of ethanol produced from arabinose varied from0.08-0.31 g h⁻¹ g⁻¹ biomass dry weight.

Initially, the constructed strain IMS0001 was able to ferment xylose(data not shown). In contrast to our expectations, the selected strainIMS0002 was not capable of fermenting xylose to ethanol (FIG. 5C). Toregain the capability of fermenting xylose, a colony of strain IMS0002was transferred to solid synthetic medium with 2% of D-xylose, andincubated in an anaerobic jar at 30° C. for 25 days. Subsequently, acolony was again transferred to solid synthetic medium with 2% ofarabinose. After 4 days of incubation at 30° C., a colony wastransferred to a shake flask containing synthetic medium with 2%arabinose. After incubation at 30° C. for 6 days, 30% of glycerol wasadded, samples were taken and stored at −80° C. A shake flask containing100 ml of synthetic medium with 2% arabinose was inoculated with such afrozen stock, and was used as preculture for an anaerobic batchfermentation on synthetic medium with 20 gl⁻¹ xylose and 20 gl¹arabinose. In FIG. 6, the fermentation profile of this batchfermentation is shown. Xylose and arabinose were consumedsimultaneously. The arabinose was completed within 70 hours, whereas thexylose was completely consumed in 120 hours. At least 250 mM of ethanolwas produced from the total of sugars, not taking into account theevaporation of the ethanol. Assuming an end biomass dry weight of 3.2gl⁻¹ (assuming a biomass yield of 0.08 g g⁻¹ sugar), the end ethanolconcentration estimated from the cumulative CO₂ production (355 mmol1⁻¹) was approximately 330 mmol 1⁻¹, corresponding to a ethanol yield of0.41 g g⁻¹ pentose sugar. In addition to ethanol, glycerol, and organicacids, a small amount of xylitol was produced (approximately 5 mM).

Selection of Strain IMS0003

Initially, the constructed strain IMS0001 was able to ferment xylose(data not shown). In contrast to our expectations, the selected strainIMS0002 was not capable of fermenting xylose to ethanol (FIG. 5C). Toregain the capability of fermenting xylose, a colony of strain IMS0002was transferred to solid synthetic medium with 2% of D-xylose, andincubated in an anaerobic jar at 30° C. for 25 days. Subsequently, acolony was again transferred to solid synthetic medium with 2% ofarabinose. After 4 days of incubation at 30° C., a colony wastransferred to a shake flask containing synthetic medium with 2%arabinose. After incubation at 30° C. for 6 days, 30% of glycerol wasadded, samples were taken and stored at −80° C.

From this frozen stock, samples were spread on solid synthetic mediumwith 2% of L-arabinose and incubated at 30° C. for several days.Separate colonies were re-streaked twice on solid synthetic medium withL-arabinose. Finally, a shake flask containing synthetic medium with 2%of L-arabinose was inoculated with a single colony, and incubated for 4days at 30° C. This culture was designated as strain IMS0003 (CBS 121879deposited at the CBS on Sep. 20, 2007). Culture samples were taken, 30%of glycerol was added and samples were stored at −80° C.

Characterization of Strain IMS0003 Growth and product formation ofstrain IMS0003 was determined during an anaerobic batch fermentation onsynthetic medium with a mixture of 30 gl⁻¹ glucose, 15 gl⁻¹ D-xylose and15 gl⁻¹ L-arabinose. The pre-culture for this anaerobic batchfermentation was prepared in a shake flasks containing 100 ml ofsynthetic medium with 2% L-arabinose, by inoculating with a −80° C.frozen stock of strain IMS0003, and incubated for 48 hours at 30° C.

In FIG. 7, the fermentation profile of a mixture of 30 gl⁻¹ glucose, 15gl⁻¹ D-xylose, and 15 gl⁻¹ L-arabinose by strain IMS0003 is shown.Arabinose consumption started after glucose depletion. Within 70 hours,the glucose, xylose and arabinose were completely consumed. Xylose andarabinose were consumed simultaneously. At least 406 mM of ethanol wasproduced from the total of sugars, not taking into account theevaporation of the ethanol. The final ethanol concentration calculatedfrom the cumulative CO₂ production was 572 mmol 1⁻¹, corresponding to anethanol yield of 0.46 g g⁻¹ of total sugar. In contrast to thefermentation of a mixture of glucose, xylose and arabinose by strainIMS0002 (FIG. 5C) or a mixed culture of strains IMS0002 and RWB218 (FIG.5D), strain IMS0003 did not produce detectable amounts of xylitol.

Tables

TABLE 1 S. cerevisiae strains used. Strain Characteristics ReferenceRWB217 MATA ura3-52 leu2-112 loxP-P_(TPI)::(−266, −1)TAL1gre3::hphMXpUGP_(TPI)- Kuyper et al. 2005a TKL1 pUGP_(TPI)-RPE1KanloxP-P_(TPI)::(−?, −1)RKI1 {p415ADHXKS, pAKX002} RWB218 MATA ura3-52leu2-112 loxP-P_(TPI)::(−266, −1)TAL1 gre3::hphMXpUGP_(TPI)- Kuyper etal. 2005b TKL1 pUGP_(TPI)-RPE1 KanloxP-P_(TPI)::(−?, −1)RKI1{p415ADHXKS1, pAKX002} RWB219 MATA ura3-52 leu2-112 loxP-P_(TPI)::(−266,−1)TAL1 gre3::hphMXpUGP_(TPI)- This work TKL1 pUGP_(TPI)-RPE1KanloxP-P_(TPI)::(−?, −1)RKI1 {p415ADHXKS1, pAKX002} RWB220 MATA ura3-52leu2-112 loxP-P_(TPI)::(−266, −1)TAL1 gre3::hphMXpUGP_(TPI)- This workTKL1 pUGP_(TPI)-RPE1 loxP-P_(TPI)::(−?, −1)RKI1 IMS0001 MATA ura3-52leu2-112 loxP-P_(TPI)::(−266, −1)TAL1 gre3::hphMXpUGP_(TPI)- This workTKL1 pUGP_(TPI)-RPE1 loxP-P_(TPI)::(−?, −1)RKI1 {pRW231, PRW243} IMS0002MATA ura3-52 leu2-112 loxP-P_(TPI)::(−266, −1)TAL1gre3::hphMXpUGP_(TPI)- This work TKL1 pUGP_(TPI)-RPE1 loxP-P_(TPI)::(−?,−1)RKI1 {pRW231, PRW243} selected for anaerobic growth on L-arabinose

TABLE 2 Plasmids used plasmid characteristics Reference pRS305Integration, LEU2 Gietz and Sugino, 1988 pAKX002 2μ, URA3,P_(TPI1)-Piromyces xylA Kuyper et al. 2003 p415ADHXKS1 CEN, LEU2,P_(ADH1)-S.cerXKS1 Kuyper et al., 2005a pRW229 integration, LEU2,P_(ADH1)-S.cerXKS1 This work pRW230 pAKX002 with P_(TDH3)-AraA This workpRW231 pAKX002 with P_(TDH3)-AraA This work and P_(HXT7)-AraD pRW243LEU2, integration, This work P_(ADH1)-ScXKS1-T_(CYC), P_(PGI1)-L.plantarumAraB-T_(ADH1)

TABLE 3 oligos used in this work Oligo DNA sequenceAraA expression cassette SpeI5′Ptdh3 5′GACTAGTCGAGTTTATCATTATCAATACTGC3′SEQ ID NO: 31 5′AraAPtdh5′CTCATAATCAGGTACTGATAACATTTTGTTTGTTTATGTGTGTTTATTC3′ SEQ ID NO: 32Ptdh5′AraA 5′GAATAAACACACATAAACAAACAAAATGTTATCAGTACCTGATTATGAG3SEQ ID NO: 33 Tadh3′AraA5′AATCATAAATCATAAGAAATTCGCTTACTTTAAGAATGCCTTAGTCAT3′ SEQ ID NO: 343′AraATadh1 5′ATGACTAAGGCATTCTTAAAGTAAGCGAATTTCTTATGATTTATGATT3′SEQ ID NO: 35 3′Tadh1SpeI 5′CACTAGTCTCGAGTGTGGAAGAACGATTACAACAGG3′SEQ ID NO: 36 AraB expression cassette SacIS′Ppgi15′CGAGCTCGTGGGTGTATTGGATTATAGGAAG3′ SEQ ID NO: 37 5′AraBPpgi15′TTGGGCTGTTTCAACTAAATTCATTTTTAGGCTGGTATCTTGATTCTA3′ SEQ ID NO: 38Ppgi5′AraB 5′TAGAATCAAGATACCAGCCTAAAAATGAATTTAGTTGAAACAGCCCAA3′SEQ ID NO: 39 Tadh3′AraB5′AATCATAAATCATAAGAAATTCGCTCTAATATTTGATTGCTTGCCCAG3′ SEQ ID NO: 403′AraBTadh1 5′CTGGGCAAGCAATCAAATATTAGAGCGAATTTCTTATGATTTATGATT3′SEQ ID NO: 41 3′Tadh1SacI 5′TGAGCTCGTGTGGAAGAACGATTACAACAGG3′SEQ ID NO: 42 AraD expression cassette SalI5′Phxt75′ACGCGTCGACTCGTAGGAACAATTTCGG3′ SEQ ID NO: 43 5′AraDPhxt5′CTTCTTGTTTTAATGCTTCTAGCATTTTTTGATTAAAATTAAAAAAACTT3′ SEQ ID NO: 44Phxt5′AraD 5′AAGTTTTTTTAATTTTAATCAAAAAATGCTAGAAGCATTAAAACAAGAAG3′SEQ ID NO: 45 Tpgi3′AraD5′GGTATATATTTAAGAGCGATTTGTTTACTTGCGAACTGCATGATCC3′ SEQ ID NO: 463′AraDTpgi 5′GGATCATGCAGTTCGCAAGTAAACAAATCGCTCTTAAATATATACC3′SEQ ID NO: 47 3′TpgiSalI 5′CGCAGTCGACCTTTTAAACAGTTGATGAGAACC3′SEQ ID NO: 48

TABLE 4 Maximum observed specific glucose and arabinose consumptionrates and ethanol production rates during anaerobic batch fermentationsof S. cerevisiae IMS0002. q_(glu) q_(ara) q_(eth,glu) q_(eth,ara)C-source g h⁻¹ g⁻¹ DW g h⁻¹ g⁻¹ DW g h⁻¹ g⁻¹ DW g h⁻¹ g⁻¹ DW 20 g l⁻¹ —0.75 ± 0.04 — 0.31 ± 0.02 arabinose 20 g l⁻¹ 2.08 ± 0.09 0.41 ± 0.010.69 ± 0.00 0.19 ± 0.00 glucose 20 g l⁻¹ arabinose 30 g l⁻¹ 1.84 ± 0.040.23 ± 0.01 0.64 ± 0.03 0.08 ± 0.01 glucose 15 g l⁻¹ xylose 15 g l⁻¹arabinose q_(glu): specific glucose consumption rate q_(ara): specificarabinose consumption rate q_(eth,glu): specific ethanol production rateduring growth on glucose q_(eth,ara): specific ethanol production rateduring growth on arabinose

REFERENCE LIST

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1. A eukaryotic cell capable of expressing the following nucleotidesequences, wherein the expression of these nucleotide sequences conferson the cell the ability to use L-arabinose and/or to convert L-arabinoseinto L-ribulose, and/or xylulose 5-phosphate and/or into a desiredfermentation product: (a) a nucleotide sequence encoding an arabinoseisomerase (araA), wherein said nucleotide sequence is selected from thegroup consisting of: i. nucleotide sequences encoding an araA, said araAcomprising an amino acid sequence that has at least 55% sequenceidentity with the amino acid sequence of SEQ ID NO:1, ii. nucleotidesequences comprising a nucleotide sequence that has at least 60%sequence identity with the nucleotide sequence of SEQ ID NO:2, iii.nucleotide sequences the complementary strand of which hybridizes to anucleic acid molecule of sequence of (i) or (ii); iv. nucleotidesequences the sequences of which differs from the sequence of a nucleicacid molecule of (iii) due to the degeneracy of the genetic code, (b) anucleotide sequence encoding a L-ribulokinase (araB), wherein saidnucleotide sequence is selected from the group consisting of: i.nucleotide sequences encoding an araB, said araB comprising an aminoacid sequence that has at least 20% sequence identity with the aminoacid sequence of SEQ ID NO:3, ii. nucleotide sequences comprising anucleotide sequence that has at least 50% sequence identity with thenucleotide sequence of SEQ ID NO:4, iii. nucleotide sequences thecomplementary strand of which hybridizes to a nucleic acid molecule ofsequence of (i) or (ii); iv. nucleotide sequences the sequences of whichdiffers from the sequence of a nucleic acid molecule of (iii) due to thedegeneracy of the genetic code, (c) a nucleotide sequence encoding anL-ribulose-5-P-4-epimerase (araD), wherein said nucleotide sequence isselected from the group consisting of: i. nucleotide sequences encodingan araD, said araD comprising an amino acid sequence that has at least60% sequence identity with the amino acid sequence of SEQ ID NO:5, ii.nucleotide sequences comprising a nucleotide sequence that has at least60% sequence identity with the nucleotide sequence of SEQ ID NO:6, iii.nucleotide sequences the complementary strand of which hybridizes to anucleic acid molecule of sequence of (i) or (ii); iv. nucleotidesequences the sequences of which differs from the sequence of a nucleicacid molecule of (iii) due to the degeneracy of the genetic code.
 2. Acell according to claim 1, wherein one, two or three of the araA, araBand araD nucleotide sequences originate from a Lactobacillus genus,preferably a Lactobacillus plantarum species.
 3. A cell according toclaim 1, wherein the cell is a yeast cell, preferably belonging to oneof the genera: Saccharomyces, Kluyveromyces, Candida, Pichia,Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.4. A cell according to claim 3, wherein the yeast cell belongs to one ofthe species: S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S.uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis.
 5. Acell according to claim 1, wherein the nucleotide sequences encoding thearaA, araB and/or araD are operably linked to a promoter that causessufficient expression of the corresponding nucleotide sequences in thecell to confer to the cell the ability to use L-arabinose and/or toconvert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/orinto a desired fermentation product.
 6. A cell according to claim 1,wherein the cell exhibits the ability to directly isomerise xylose intoxylulose.
 7. A cell according to claim 6, wherein the cell comprises agenetic modification that increases the flux of the pentose phosphatepathway.
 8. A cell according to claim 6, wherein the geneticmodification comprises overexpression of at least one gene of thenon-oxidative part of the pentose phosphate pathway.
 9. A cell accordingto claim 8, wherein the gene is selected from the group consisting ofthe genes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase.
 10. A cell according toclaim 8, wherein the genetic modification comprises overexpression of atleast the genes coding for a transketolase and a transaldolase.
 11. Acell according to claim 8, wherein the cell further comprises a geneticmodification that increases the specific xylulose kinase activity.
 12. Acell according to claim 11, wherein the genetic modification comprisesoverexpression of a gene encoding a xylulose kinase.
 13. A cellaccording to claim 8, wherein the gene that is overexpressed isendogenous to the cell.
 14. A cell according to claim 5, wherein thecell comprises a genetic modification that reduces unspecific aldosereductase activity in the cell.
 15. A cell according to claim 14,wherein the genetic modification reduces the expression of, orinactivates a gene encoding an unspecific aldose reductase.
 16. A cellaccording to claim 15, wherein the gene is inactivated by deletion of atleast part of the gene or by disruption of the gene.
 17. A cellaccording to claim 14, wherein the expression of each gene in the cellthat encodes an unspecific aldose reductase is reduced or inactivated.18. A cell according to claim 1, wherein the fermentation product isselected from the group consisting of ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin.19. A nucleic acid construct comprising a nucleic acid sequence encodingan araA, a nucleic acid sequence encoding an araB and/or a nucleic acidsequence encoding an araD all as defined in claim
 1. 20. A process forproducing a fermentation product selected from the group consisting ofethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, aceticacid, succinic acid, citric acid, malic acid, fumaric acid, an aminoacid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactamantibiotic and a cephalosporin, whereby the process comprises: (a)fermenting a medium containing a source of arabinose and optionallyxylose with a modified cell as defined in claim 1, whereby the cellferments arabinose and optionally xylose to the fermentation product;and optionally, (b) recovering the fermentation product.
 21. A processfor producing a fermentation product selected from the group consistingof ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, aceticacid, succinic acid, citric acid, malic acid, fumaric acid, an aminoacid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactamantibiotic and a cephalosporin, wherein the process comprises: (a)fermenting a medium containing at least a source of L-arabinose and asource of xylose with a cell as defined in claim 1 and a cell able touse xylose and/or exhibiting the ability to directly isomerise xyloseinto xylulose, whereby each cell ferments L-arabinose and/or xylose tothe fermentation product; and optionally, (b) recovering thefermentation product.
 22. A process according to claim 20, wherein themedium also contains a source of glucose.
 23. A process according toclaim 20, wherein the fermentation product is ethanol.
 24. A processaccording to claim 23, wherein the volumetric ethanol productivity is atleast 0.5 g ethanol per litre per hour.
 25. A process according to claim23, wherein the ethanol yield is at least 30%.
 26. A process accordingto claim 20, wherein the process is anaerobic.
 27. A process accordingto claim 20, wherein the process is aerobic, preferably performed underoxygen limited conditions.